CN101580538B

CN101580538B - WT1-origin HLA-DR-binding antigen peptide

Info

Publication number: CN101580538B
Application number: CN2009101426956A
Authority: CN
Inventors: 杉山治夫
Original assignee: International Institute of Cancer Immunology Inc
Current assignee: International Institute of Cancer Immunology Inc
Priority date: 2003-11-05
Filing date: 2004-11-04
Publication date: 2013-06-19
Anticipated expiration: 2024-11-04
Also published as: CY1112585T1; KR101522079B1; CA2544214C; SI2071028T1; JP4621142B2; WO2005045027A1; CN100513563C; EP2071028A2; KR20120067374A; KR20130062368A; US20080070835A1; KR20060123756A; EP1696027A1; EP2071028B1; KR101213015B1; CN1902313A; JPWO2005045027A1; ES2378264T3; PL2071028T3; EP2071028A3

Abstract

The present invention provides a WT1-derived HLA-DRB1*0405-binding antigen peptide, a polynucleotide encoding said peptide, a helper T cell inducer comprising said peptide or polynucleotide, and the like. It is related to a partial peptide consisting of 10 - 25 contiguous amino acids in the amino acid sequence of human WT1 shown in SEQ ID NO: 1, which binds to HLA-DRB1*0405 and induces helper T cells, a polynucleotide encoding said peptide, or a helper T cell inducer comprising said peptide or polynucleotide.

Description

The HLA-DR-conjugated antigen peptide that WT1 is derivative

The application is dividing an application of following application: November 04 2004 applying date, application number 200480039886.5 (PCT/JP2004/016336), title " the HLA-DR-conjugated antigen peptide that WT1 is derivative ".

Technical field

The present invention relates to the derivative HLA-DRB1*0405-conjugated antigen peptide of WT1.

Background technology

Identified that WT1 gene (Wilms ' oncogene 1) is that Wilms ' tumour is one of Children with Renal tumour Disease-causing gene (Cell 60:509,1990, Nature 343:774,1990).WT1 genes encode transcription factors WT1, this factor play an important role in all multi-process such as cell proliferation, differentiation, apoptosis and tissue development (Int.Rev.Cytol.181:151,1998).It is a kind of tumor suppressor gene that initial WT1 gene is described to.But, follow-up study disclosed the WT1 gene leukemia and comprise lung cancer and the multiple solid tumor of mammary cancer in high expression level, show that the WT1 gene more properly bringing into play the carcinogenesis that promotes growth of cancers.In addition, leukemia and the solid tumor cell of having induced peptide-specificity cell toxicity T-lymphocyte (CTLs) and having killed endogenous expression WT1 have been confirmed when when the derivative peptide of external use WT1-stimulates the peripheral blood lymphocytes of HLA-A*0201 or the HLA-A*2402 positive.These results confirm that WT1 is the target molecule (Int.J.Hematol76:127,2002) of immunotherapy for cancer expectation.

The existence of already having reported the helper T cell that is specific to cancer antigen is necessary (Cancer.Res.62:6438,2002) for effectively inducing CTLs.

When on its identification antigen presenting cell when MHC II quasi-molecule and antigenic peptide complexes, induce (causing propagation) and activate helper T cell (CD4 positive T cell).The helper T cell that activates produces such as the cytokine of IL-2, IL-4, IL-5, IL-6 and/or Interferon, rabbit and mediates growth, differentiation and the maturation of B cell.The helper T cell of this activation also works to promote growth, differentiation and the maturation such as other T cell subsets of Tc and TD cell.Therefore, the helper T cell of this activation can pass through promote the growth of B and T cell and activate with activated immune system.Therefore, think under the impact of MHC II class conjugated antigen peptide (also referred to as " auxiliary peptide "), it is useful strengthening the helper T cell function, taken this to increase effect (the effect) (J.Immunother. of cancer vaccine in immunotherapy for cancer (cancer vaccine therapy), 24:195,2001).

With regard to the WT1-derived peptide, only known have a kind of antigen Toplink in conjunction with MHC II quasi-molecule hypotype, i.e. HLA-DRB1*0401 (Cancer Immunol.Immunother.51:271,2002).Not having can be in conjunction with the report of the WT1-derived peptide of different subtype.

Summary of the invention

The purpose of this invention is to provide the derivative HLA-DRB1*0405-conjugated antigen peptide of WT1, and this peptide is as the application of cancer vaccine effect toughener (a kind of reagent for strengthening the cancer vaccine effect).

Derivative to the WTI-with the activity that strengthens cancer vaccine effect (effect) in conjunction with MHC II class antigen and in the immunotherapy for cancer antigen peptide (" auxiliary peptide ") of the inventor has been carried out thorough research.As a result, the inventor has found that first WT1 contains the antigen peptide part, and it has the activity that HLA-DRB1*0405 in many MHC II class subclass is combined and is induced helper T cell.This discovery has promoted the development of new treatment, is namely induced in the positive cancer patients of HLA-DRB1*0405-by the method and strengthens WT1-specificity helper T cell.

Recently have research to disclose existing non-kind is the auxiliary peptide of selectivity (promiscuous), it is to be combined and to induce auxiliary peptide (the British J.Cancer of complementary CD4 positive T cell with multiple HLA II quasi-molecule, 85 (10), p1527-1534 (2001); J.Immunol., 169, p557-565 (2002)).For illustrating WT1 _332-347Whether be that potential non-kind is the auxiliary peptide of selectivity, the inventor is to this WT1 as above-mentioned HLA-DRB1*0405-conjugated antigen peptide (auxiliary peptide) _332-347Be studied.As a result, it is the auxiliary peptide of selectivity that described peptide is proved to be non-kind, and it not only is combined with the HLA-DRB1*0405 molecule, also is combined with the HLA-DRB1*1502 molecule.Therefore, WT1 of the present invention _332-347Peptide is a kind of auxiliary peptide that can be applicable to have HLA-DRB1*1502 and HLA-DRB1*0405 patient.The inventor also found first and can be combined with the HLA-DRB1*1502 of one of multiple MHC II class subclass, and the antigen peptide part of inducing the WT1 of helper T cell to contain.

Set up the present invention on the basis of these discoveries.

Summary of the invention

The present invention includes as follows.

(1) a kind of by 10-25 peptide that continuous amino acid forms in the people WT1 aminoacid sequence as shown in SEQ ID NO:1, it is in conjunction with HLA-DRB1*0405 and induce helper T cell.

(2) peptide of above-mentioned (1), it comprises the aminoacid sequence as shown in as arbitrary in SEQ ID NOS:2-23.

(3) peptide of above-mentioned (2), it comprises the aminoacid sequence as shown in SEQ ID NO:24.

(4) a kind of 10-25 amino acid whose peptide, its amino-acid residue of the 1st, 4,6 and/or 9 that comprises the aminoacid sequence as shown in as arbitrary in SEQ ID NOS:2-23 is by the aminoacid sequence of different aminoacids gained that residue replaces, and it is in conjunction with HLA-DRB1*0405 and induce helper T cell.

(5) peptide of above-mentioned (4), it comprises the selected aminoacid sequence from gained that following amino acid whose amino-acid residue replaces of the amino-acid residue of the 1st, 4,6 and/or 9 of the aminoacid sequence as shown in as arbitrary in SEQ ID NOS:2-23:

For the 1st, phenylalanine, tyrosine, tryptophane, α-amino-isovaleric acid, Isoleucine, leucine and methionine(Met);

For the 4th, α-amino-isovaleric acid, Isoleucine, leucine, methionine(Met), aspartic acid and L-glutamic acid;

For the 6th, l-asparagine, Serine, Threonine, glutamine, Methionin and aspartic acid; And

For the 9th, aspartic acid, L-glutamic acid and glutamine.

(6) peptide of above-mentioned (5), it comprises the selected aminoacid sequence from gained that following amino acid whose amino-acid residue replaces of the amino-acid residue of the 3rd, 6,8 and/or 11 of the aminoacid sequence as shown in SEQ ID NO:24:

For the 3rd, phenylalanine, tryptophane, α-amino-isovaleric acid, Isoleucine, leucine and methionine(Met);

For the 6th, α-amino-isovaleric acid, Isoleucine, methionine(Met), aspartic acid and L-glutamic acid;

For the 8th, l-asparagine, Serine, Threonine, glutamine, Methionin and aspartic acid; And

For the 11st, aspartic acid, L-glutamic acid and glutamine.

(7) a kind of by 10-25 peptide that continuous amino acid forms in the people WT1 aminoacid sequence as shown in SEQ ID NO:1, it is in conjunction with HLA-DRB1*1502 and induce helper T cell.

(8) peptide of above-mentioned (7), it comprises the aminoacid sequence as shown in as arbitrary in SEQ ID NOS:46-56.

(9) peptide of above-mentioned (7), it comprises the aminoacid sequence as shown in SEQ ID NO:24.

(10) a kind of peptide that comprises described peptide as arbitrary in above-mentioned (1)-(9) and cancer antigen peptide.

(11) polynucleotide of a kind of coding as the arbitrary described peptide in above-mentioned (1)-(10).

(12) a kind of expression vector that comprises as above-mentioned (11) described polynucleotide.

(13) a kind of cell that contains as above-mentioned (12) described expression vector.

(14) a kind of production as the method for the arbitrary described peptide in above-mentioned (1)-(10), it is included under the condition that this Toplink expresses and cultivates as above-mentioned (13) described cell.

(15) antibody of the arbitrary described peptide of a kind of specific binding such as above-mentioned (1)-(9).

(16) a kind of pharmaceutical composition, it comprises described peptide as arbitrary in above-mentioned (1)-(10), as above-mentioned (12) described expression vector, or as above-mentioned (13) described cell, and pharmaceutically acceptable carrier.

(17) pharmaceutical composition of above-mentioned (16), it is a kind of cancer therapy and preventive.

(18) pharmaceutical composition of above-mentioned (16), it is a kind of helper T cell inductor, and it comprises described peptide as arbitrary in above-mentioned (1)-(9); (12) described expression vector relevant with described peptide as arbitrary in above-mentioned (1)-(9); Or (13) described cell relevant with described peptide as arbitrary in above-mentioned (1)-(9), and pharmaceutically acceptable carrier.

(19) pharmaceutical composition of above-mentioned (16), it is a kind of cancer vaccine effect toughener, and it comprises described peptide as arbitrary in above-mentioned (1)-(9); (12) described expression vector relevant with described peptide as arbitrary in above-mentioned (1)-(9); Or (13) described cell relevant with described peptide as arbitrary in above-mentioned (1)-(9), and pharmaceutically acceptable carrier.

(20) pharmaceutical composition of above-mentioned (16), it is a kind of cancer therapy or preventive, and it comprises as above-mentioned (10) described peptide; With (12) described expression vector as relevant in above-mentioned (10) described peptide; Or with (13) described cell as relevant in above-mentioned (10) described peptide, and pharmaceutically acceptable carrier.

(21) described peptide as arbitrary in above-mentioned (1)-(10), as above-mentioned (12) described expression vector or as the application of above-mentioned (13) described cell in making cancer therapy or preventive.

(22) a kind of method for the treatment of or preventing cancer, it comprise to required patient use described peptide as arbitrary in above-mentioned (1)-(10), as above-mentioned (12) described expression vector or as above-mentioned (13) described cell.

(23) a kind of pharmaceutical composition, it is combined by described peptide as arbitrary in above-mentioned (1)-(9) and a kind of cancer antigen peptide.

(24) pharmaceutical composition of above-mentioned (23), it is used for the treatment of or preventing cancer.

(25) a kind of being used for the treatment of or the test kit of preventing cancer, it comprises a kind of pharmaceutical composition that comprises as the arbitrary described peptide in above-mentioned (1)-(9) and pharmaceutically acceptable carrier, and a kind of pharmaceutical composition that comprises cancer antigen peptide and pharmaceutically acceptable carrier.

(26) described peptide as arbitrary in above-mentioned (1)-(9) and cancer antigen peptide is combined in the application of making in cancer therapy or preventive.

(27) a kind of method for the treatment of or preventing cancer, it comprises uses described peptide as arbitrary in above-mentioned (1)-(9) and cancer antigen peptide to required patient.

The invention provides the derivative HLA-DRB1*0405-conjugated antigen peptide of a kind of WT1, this peptide of encoding polynucleotide, comprise helper T cell inductor (" helper T cell inductor ") of described peptide or polynucleotide etc.Helper T cell inductor of the present invention is used as cancer vaccine effect toughener.Cancer vaccine effect toughener of the present invention is applicable to many HLA-DRB1*0405-positive patients, and is particularly useful for the effect that strengthens the WT1 vaccine.

Description of drawings

Fig. 1 has shown the derivative WT1 of WT1- _332-347The test-results that the CD4 positive T cell (helper T cell) that peptide stimulates and various dendritic cell are replied.In the figure, " untreated " expression is not to using replying of dendritic cell that peptide stimulates; " PHA " expression wherein replaces with PHA the test-results that dendritic cell are processed the CD4 positive T cell." WT1 _172-186Stimulate " represent WT1 _172-186Replying of the dendritic cell that peptide stimulates, " WT1 _225-243Stimulate " represent WT1 _225-243Replying of the dendritic cell that peptide stimulates, and " WT1 _332-347Stimulate " represent WT1 _332-347Replying of the dendritic cell that peptide stimulates.The longitudinal axis represent that the CD4 positive T cell takes in [ ³H]-amount (cpm) of thymidine.

Fig. 2 has shown the WT1 that G2 clone is derivative to WT1- _332-347The test-results that the dendritic cell that peptide stimulates are replied.In the figure, the result that the dendritic cell do not carry out peptide and stimulate obtain is used in " untreated " expression; And " WT1 _332-347Pulse " expression use WT1 _332-347The result that the dendritic cell that stimulate obtain.The longitudinal axis represent that G2 clone takes in [ ³H]-amount (cpm) of thymidine.

Fig. 3 has shown the test-results of G2 clone to B-LCL (+) cell response of expression WT1 gene.In the figure, the result that " B-LCL (-) " expression uses B-LCL (-) cell do not express the WT1 gene to obtain, the result that B-LCL (+) cell of WT1 gene obtains use is expressed in " B-LCL (+) " expression, and " B-LCL (+)+anti--HLA-DR antibody " expression result of using B-LCL (+) cell of anti--HLA-DR antibody treatment to obtain.The longitudinal axis represent that G2 clone takes in [ ³H]-amount (cpm) of thymidine.

Fig. 4 has shown the WT1 that E04.1 clone is derivative to WT1- _332-347The test-results that the dendritic cell that peptide stimulates are replied.In the figure, the result that "-" expression uses the dendritic cell do not carry out peptide and stimulate to obtain, and WT1 is used in " 332 " expression _332-347The result that the dendritic cell that peptide stimulates obtain.The longitudinal axis represent that E04.1 clone takes in [ ³H]-amount (cpm) of thymidine.

Fig. 5 has shown the WT1 of E04.1 clone to first deriving with WT1- _332-347Peptide stimulates the test-results of replying with the irritation cell (stimulated cells) that various resisting-the HLA inhibiting antibody is processed again.The irritation cell that uses is B-LCL (-) cell, and it is a kind of foundation from the B clone to healthy volunteer's blood of the HLA-DRB1*0405 positive, as shown in following examples 3 herein.In the figure, the result that "-" expression uses the irritation cell do not carry out peptide and stimulate to obtain, and WT1 is used in " 332 " expression _332-347The result that the irritation cell that peptide stimulates obtains.In addition, WT1 is used in " 332+ α-I class " expression _332-347The result that the irritation cell that peptide and anti--HLA-I antibody-like are processed obtains, WT1 is used in " 332+ α-DR " expression _332-347The result that the irritation cell of peptide and anti--HLA-DR antibody treatment obtains, WT1 is used in " 332+ α-DQ " expression _332-347The result that the irritation cell of peptide and anti--HLA-DQ antibody treatment obtains.In addition, WT1 is used in " 332+mIgG " expression _332-347Peptide and as the inhibiting antibody negative control anti--result that the irritation cell of mouse-IgG antibody treatment obtains.The longitudinal axis represent that E04.1 clone takes in [ ³H]-amount (cpm) of thymidine.

Fig. 6 has shown the WT1 of E04.1 clone to deriving with WT1- _332-347The test-results that the HLA-DRB1*0405-positive or negative PBMC that peptide stimulates replys.In the figure, the result that "-" expression uses the PBMC do not carry out peptide and stimulate to obtain, and WT1 is used in " 332 " expression _332-347The result that the PBMC that stimulates obtains.The HLA-DRB1 genotype of various donors is as follows.Donor 1 (HLA-DRB1*0405/0803), donor 2 (HLA-DRB1*0405/0101), donor 3 (HLA-DRB1*0101/1001), and donor 4 (HLA-DRB1*1201/0802).The longitudinal axis represent that E04.1 clone takes in [ ³H]-amount (cpm) of thymidine.

Fig. 7 has shown the test-results of E04.1 clone to B-LCL (+) cell response of expression WT1 gene.In the figure, the result that " B-LCL (-) " expression uses B-LCL (-) gene do not express the WT1 gene to obtain as irritation cell, and " B-LCL (+) " expression result of using B-LCL (+) gene of expressing the WT1 gene to obtain as irritation cell.The longitudinal axis represent that E04.1 clone takes in [ ³H]-amount (cpm) of thymidine.

Fig. 8 has shown the test-results of E04.1 clone to replying with the dendritic cell of apoptosis-induced B-LCL (+) cytositimulation.In the figure, the result that the dendritic cell of WT1 gene and apoptosis-induced B-LCL (+) cytositimulation obtain use is expressed in " B-LCL (+) of apoptosis " expression, and " B-LCL (-) of apoptosis " expression result of using the dendritic cell of not expressing WT1 gene and apoptosis-induced B-LCL (-) cytositimulation to obtain.In addition, the result that obtains by cultivating altogether E04.1 cell and dendritic cell of " E04.1+ " expression, and the result of " E04.1-" expression by not obtaining with the E04.1 co-culture of cells.The longitudinal axis represent that E04.1 clone takes in [ ³H]-amount (cpm) of thymidine.

Fig. 9 has shown the WT1 of E04.1 clone to deriving with WT1- _332-347The dendritic cell that peptide stimulates are replied the test-results that middle cytokine produces.In the figure, the result that "-" expression uses the dendritic cell do not carry out peptide and stimulate to obtain, and WT1 is used in " 332 " expression _332-347The result that the dendritic cell that peptide stimulates obtain.The longitudinal axis represents to show the per-cent (%) of the E04.1 cell that produces IL-4 (blank) or IFN-γ (packing).

Figure 10 has shown the result of using the E04.1 clone of anti-CD 4 antibodies and anti--CXCR3 antibody staining by flow cytometry analysis.In the figure, transverse axis and the longitudinal axis represent respectively the cell to CD4 and the CXCR3 positive.Cell per-cent to CD4 and the CXCR3 positive is 90.1%..

Figure 11 has shown the derivative WT1 of WT1- _332-347Peptide is induced the test-results of impact on WT1-specific CTL s.At (A) WT1 _235-243Peptide, (B) WT1 _235-243Peptide+WT1 _332-347Peptide, (C) WT1 _235-243Peptide+E04.1 cell and (D) WT1 _235-243Peptide+WT1 _332-347Under peptide+E04.1 cytositimulation condition, cultivate from healthy volunteer's (HLA-A*2402/1101, DRB1*0405-0803) PBMCs 7 days.Then, with flow cytometer to half recovery cell analysis to obtain WT1 _235-243The per-cent of-specific CTL precursor.Transverse axis and the longitudinal axis represent respectively CD8 and WT1 _235-243The per-cent of peptide/HLA-A*2402 positive cell.

Figure 12 has shown the derivative WT1 of WT1- _332-347The test-results of peptide on WT1-specific CTL s activation impact.Use WT1 _235-243Peptide stimulates in the test that Figure 11 mentions second half recovery cell 6 hours, and in born of the same parents, IFN-γ is colored.The longitudinal axis and transverse axis represent respectively the cell to IFN-γ in born of the same parents and anti--mouse IgG antibody positive.The figure illustrates use (E) WT1 _235-243Peptide, (F) WT1 _235-243Peptide+WT1 _332-347Peptide, (G) WT1 _235-243Peptide+E04.1 cell and (H) WT1 _235-243Peptide+WT1 _332-347The result of peptide+E04.1 cytositimulation

Figure 13 has shown the derivative WT1 of WT1- _332-347The test-results that the CD4 positive T cell that peptide stimulates and various dendritic cell are replied.In the figure, the result that "-" expression uses the dendritic cell do not carry out peptide and stimulate to obtain, WT1 is used in " 332 " expression _332-347The result that the dendritic cell that peptide stimulates obtain, WT1 is used in " 172 " expression _172-186The result that the dendritic cell that peptide stimulates obtain, and WT1 is used in " 225 " expression _225-243The result that the dendritic cell that peptide stimulates obtain.The longitudinal axis represent that the CD4 positive T cell takes in [ ³H]-amount (cpm) of thymidine.Symbol " * * " and " n.s. " represent that respectively in test group, difference is statistically significant or inapparent.

Figure 14 has shown the derivative WT1 with WT1- _332-347The result that peptide stimulates the t cell responses spectrum (repertoire) of CD4 positive T cell to analyze.With the different antibodies staining cell that is specific to the various V β of TCR chain and analyze by flow cytometer.In lastrow figure, the cell mass in four subregion lower right-most portion represents V β 3-positive cell.In next line figure, the cell mass in four subregion lower right-most portion represents V β 20-positive cell.

Figure 15 has shown E15.1 clone or the E15.2 clone test-results of replying from body PBMCs to stimulating with the derivative WT1332-347 peptide of WT1-.In the figure, the result that obtains from body PBMCs do not carry out that peptide stimulates is used in "-" expression, and WT1 is used in " 332 " expression _332-347The result that obtains from body PBMCs that peptide stimulates.The longitudinal axis represent that different clones take in [ ³H]-amount (cpm) of thymidine.A) and B) shown respectively the result of using E15.1 clone and E15.2 clone to obtain.In symbol " * * " expression test group, difference is statistically significant.

Figure 16 has shown that E15.2 clone replys from body PBMCs the test-results that middle cytokine produces to what stimulate with the derivative WT1332-347 peptide of WT1-.In the figure, the result that "-" expression uses the dendritic cell do not carry out peptide and stimulate to obtain, and WT1 is used in " 332 " expression _332-347The result that obtains from body PBMCs that peptide stimulates.The longitudinal axis represents to show the per-cent (%) of the E15.2 cell that produces IL-4 (blank) or IFN-γ (packing).

What Figure 17 had shown that the derivative WT1332-347 peptide of WT1-stimulates replys the test-results of Relations Among from body PBMCs concentration and E15.2 clone.The longitudinal axis represent that E15.1 clone takes in [ ³H]-amount (cpm) of thymidine.Transverse axis represents the concentration from body PBMC that the WT1332-347 peptide stimulates.

Figure 18 has shown the test-results that E15.2 clone is replied the PBMCs of the HLA-DRB1*1502 positive or negative that stimulates with the derivative WT1332-347 peptide of WT1-.In the figure, the result that "-" expression uses the PBMCs do not carry out the WT1332-347 peptide and stimulate to obtain, and the result that obtains of " 332 " expression PBMCs of using this peptide to stimulate.A) shown result and the B that uses the PBMCs from the positive healthy volunteer of HLA-DRB1*1502-to obtain) shown the result of using the PBMCs from the negative healthy volunteer of HLA-DRB1*1502-to obtain.The longitudinal axis represent that E15.2 clone takes in [ ³H]-amount (cpm) of thymidine.Symbol " * * " and " n.s. " represent that respectively in test group, difference is statistically significant or inapparent.

Embodiment

The invention provides a kind ofly by 10-25 peptide that continuous amino acid forms in the people WT1 aminoacid sequence as shown in SEQ ID NO:1, described peptide is in conjunction with HLA-DRB1*0405 and induce helper T cell.The present invention includes wherein N-end and/or the adorned peptide of C-terminal amino acid residue or reformed those peptides of particular amino acid residue wherein.

Hereinafter, " induce the peptide peptide of CD4 positive T cell (or induce) of helper T cell " and can be described as " auxiliary peptide ".

People WT1 aminoacid sequence as shown in SEQ ID NO:1 is known sequence, is described in Cell, 60:509,1990 and ncbi database (accession number XP_034418 and P19544) in.

Peptide of the present invention is part (partial) peptide that 10-25 continuous amino acid in a kind of people WT1 aminoacid sequence by being present in as shown in SEQ ID NO:1 forms.The definition of being somebody's turn to do " 10-25 amino acid " is based on the peptide that usually is comprised of 10-25 amino acid to have in conjunction with active this fact (Immunogenetics of MHC II class, 41:178-228,1995, Biochimica et Biophysica Acta 1316,85-101 (1996), Immunology, 96,1-9 (1999), Peptides, Vol.19,179-198 (1998), Immunobiology, the 5th edition, 116-117, Garland Publishing (2001)).Preferred peptide is that those are comprised of 13-17 continuous amino acid in people WT1 aminoacid sequence.

Whether peptide of the present invention is by the synthetic peptide (candidate's peptide) that is comprised of 10-25 continuous amino acid in the people WT1 aminoacid sequence as shown in SEQ ID NO:1, and measure described peptide and can be combined with HLA-DRB1*0405 and induce helper T cell and identified.

Can according in the chemistry of peptides field usually method used carry out the synthetic of peptide.Such method is found in document, comprises Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol.2 Academic Press Inc., New York, 1976; PeptideSynthesis, Maruzen, Inc., 1975; Pepteide-Gosei no Kiso to Jikken, Maruzen, Inc.1985; And Iyakuhin no Kaihatsu (Zoku), Vol.14, Peptide Synthesis, Hirokawa-syoten, 1991.

Use is described in, for example, method in Cancer.Immunol.Immunother.51:271 (2002), be described in method in embodiment or method as described below, can detect candidate's peptide and whether can and induce helper T cell in conjunction with HLA-DRB1*0405.

Specifically, by separating periphery blood monocytic cell (PBMCs) from people's object of the HLA-DRB1*0405 positive and remove not attached cell, preparation dendritic cell (attached cell).Respectively, prepare helper T cell (CD4 positive T cell) by using Ficoll-Paque (Ficoll-Paque) to carry out density gradient centrifugation etc. from the positive object of identical HLA-DRB1*0405-.

Cultivate above-mentioned dendritic cell after having added candidate's peptide, and further cultivate with above-mentioned helper T cell.Then the dendritic cell that reclaim helper T cell and stimulate with candidate's peptide stimulate several times in a similar manner.By measuring, for example, the cytokine of the growth activity of (1) helper T cell or (2) helper T cell produces active, might assess helper T cell and whether be induced (activation) in replying the peptide stimulation.Specifically, growth activity (1) can be by measuring the check that measures of [3H]-thymidine that helper T cell takes in.Cytokine produces active (2) and can be checked by the amount such as the cytokine of IFN-γ that enzyme-linked immunosorbent assay (ELISA) or similar approach measure that the helper T cell that activates produce.

Aminoacid sequence in conjunction with MHC I class or MHC II quasi-molecule and the antigen peptide of presenting is followed a certain rule (binding motif).Its for play an important role in MHC I quasi-molecule is combined at the terminal amino acid residue in conjunction with two ends of the peptide of MHC I quasi-molecule; But, do not have such amino acid at the arbitrary end in conjunction with the peptide of MHC II quasi-molecule, and this end amino acid is not combined with MHC II quasi-molecule.Such peptide is more suitable for and is vertically fixed in (being fixed on) peptide binding groove groove.In the peptide binding groove groove peptide fixing can by combination consists of the amino acid whose side chain of this peptide and peptide binding groove groove and in conjunction with this peptide main chain and in the complete peptide binding groove groove of MHC II quasi-molecule the good amino acid whose side chain of preservation with acquisition.The peptide binding groove groove has little or large pocket, and depends on the amino acid polymorphism that MHC II quasi-molecule has in the amino-acid residue that consists of this pocket.

The X-radiocrystallgraphy that obtains up to now disclosed with the 1st, 4,6 and 9 side chain of locating amino-acid residue of the MHC II class-binding peptide of the minimum of these binding pocket joinings.

Be common in from the not homoallelic MHC II quasi-molecule amino-acid residue pattern in the peptide of combination separately by analysis, can assess the amino acid motif of the peptide of being combined with the pocket of peptide binding groove groove.Consider, because approximately 9 amino acid whose peptides have the above-mentioned motif of suitable peptide binding groove groove, this is fit to carry out in this manner, and namely two ends stretch out from two sites of groove, basically do not limit the length of the peptide that can be combined with MHC II quasi-molecule.But, in many cases, will long peptide be cut into the peptide (Immunobiology, the 5th edition, 116-117, Garland Publishing (2001)) of 13-17 amino acid long by peptase.

Just has HLA-DRB1*0405 in conjunction with regard to the peptide of activity, at the HLA that is formed by 9 amino acid (MHC)-estimate to have following systematicness (motif) (Immunogenetics in conjunction with the amino acid at the 1st, 4,6 and 9 place in territory, 41:178-228,1995, Biochimica etBiophysica Acta 1316,85-101 (1996)).

The 1st: phenylalanine (F), tyrosine (Y), tryptophane (W), α-amino-isovaleric acid (V), Isoleucine (I), leucine (L) and methionine(Met) (M)

The 4th: α-amino-isovaleric acid (V), Isoleucine (I), leucine (L), methionine(Met) (M), aspartic acid (D) and L-glutamic acid (E)

The 6th: l-asparagine (N), Serine (S), Threonine (T), glutamine (Q), Methionin (K) and aspartic acid (D)

The 9th: aspartic acid (D), L-glutamic acid (E) and glutamine (Q).

Recently, use the software (Propred, Bioinformatics 17:1236,2001) of MHC II class binding peptide might search the peptide sequence that expection is combined with MHC II class antigen.

The present invention is based on and finds that first WT1 (SEQ ID NO:1) contains in conjunction with HLA-DRB1*0405 (a kind of MHC II class) and induces the antigen peptide part of helper T cell.The HLA-DRB1*0405-that infers in described WT1 aminoacid sequence comprises in conjunction with the example of nine amino acid part the nine amino acid part that WT1 is derivative, as shown in SEQ ID NOS:2-23.Therefore, as the particular of peptide of the present invention, the invention provides a kind of aminoacid sequence as shown in as arbitrary in SEQ ID NOS:2-23 that comprises, in conjunction with HLA-DRB1*0405 and induce the peptide of helper T cell.

Be the partial peptide of WT1 and comprise the aminoacid sequence as shown in as arbitrary in SEQ ID NOS:2-23 at described peptide, in conjunction with HLA-DRB1*0405 and induce under the peptide condition of helper T cell, do not limit the length of above-mentioned peptide.As above, approximately 9 the amino acid whose peptides with binding motif can be suitable for the peptide binding groove groove, and its two ends stretch out from the both sides of groove, therefore basically do not limit the length of the peptide that can be combined with MHC II quasi-molecule.But long peptide is generally peptase and cuts, and has reported that up to now MHC II class binding peptide length is about 10-25 amino acid (Immunogenetics, 41:178-228,1995, Biochimica et BiophysicaActa 1316,85-101 (1996), Immunology, 96,1-9 (1999), Peptides, Vol.19,179-198 (1998), Immunobiology, the 5th edition, 116-117, GarlandPublishing (2001)).Consider this point, peptide of the present invention preferably is comprised of about 10-25 amino acid, more preferably is comprised of about 13-17 amino acid.

Therefore, the example of preferred embodiment that comprises the peptide of the present invention of the aminoacid sequence as shown in as arbitrary in SEQ ID NOS:2-23 comprise by 10-25 amino acid form (preferably, formed by 13-17 amino acid) the derivative partial peptide of WT1, it comprises the aminoacid sequence as shown in as arbitrary in SEQ ID NOS:2-23, and has in conjunction with HLA-DRB1*0405 and induce the activity of helper T cell.

The example of preferred embodiment comprise by 10-25 amino acid form (preferably, formed by 13-17 amino acid) the derivative partial peptide of WT1, it comprises the aminoacid sequence as shown in SEQ ID NO:12, and has in conjunction with HLA-DRB1*0405 and induce the activity of helper T cell.Still the example of preferred embodiment comprise by 16-25 amino acid form (preferably, formed by 16-17 amino acid) the derivative partial peptide of WT1, it comprises the aminoacid sequence as shown in SEQ ID NO:24, and has in conjunction with HLA-DRB1*0405 and induce the activity of helper T cell.Aminoacid sequence as shown in SEQ ID NO:24 represents the 16 amino acid whose partial peptides that WT1 is derivative, and comprises the aminoacid sequence as shown in SEQ ID NO:12.

The peptide that the most preferred embodiment is comprised of the aminoacid sequence as shown in SEQ ID NO:24.

Peptide of the present invention can carry out suitable change in the scope that keeps activity.As used herein " change " of amino-acid residue refer to replacement, disappearance and/or the interpolation (described interpolation is included in N-and/or the amino acid whose interpolation in C-end of peptide) of amino-acid residue.The replacement of amino-acid residue is preferred.When change related to the amino-acid residue replacement, under the condition of auxiliary peptide activity keeping, the amino-acid residue of the arbitrary number of any position can be substituted.But, because usually be about 10-25 amino acid in conjunction with the peptide of HLA II quasi-molecule, preferably relate to 1 to several amino acid so change.

When changing amino-acid residue by replacement, the amino-acid residue at the 1st, 4,6 and/or 9 place that preferably has the nine amino acid peptide of HLA-DRB1*0405 binding motif structure is substituted.

The object lesson of the relevant peptide that replaces of the present invention comprises 10-25 amino acid whose peptide, its the 1st, 4,6 and/or 9 of comprising the aminoacid sequence as shown in as arbitrary in SEQ ID NOS:2-23 locates amino-acid residue and is replaced by the different aminoacids residue, and is combined with HLA-DRB1*0405 and induces the aminoacid sequence of helper T cell.

Preferred example comprises 10-25 amino acid whose peptide, its the 1st, 4,6 and/or 9 of comprising the aminoacid sequence as shown in as arbitrary in SEQ ID NOS:2-23 locates amino-acid residue for being selected from following amino acid: for the 1st, and phenylalanine, tyrosine, tryptophane, α-amino-isovaleric acid, Isoleucine, leucine and methionine(Met); For the 4th, α-amino-isovaleric acid, Isoleucine, leucine, methionine(Met), aspartic acid and L-glutamic acid; For the 6th, l-asparagine, Serine, Threonine, glutamine, Methionin and aspartic acid; And for the 9th, the amino-acid residue of aspartic acid, L-glutamic acid and glutamine replaces, and in conjunction with HLA-DRB1*0405 and induce the aminoacid sequence of helper T cell.

For example, can carry out the 1st, 4,6 and/or 9 replacement of locating amino-acid residue, be used for improvement HLA-DRB1*0405 in conjunction with active or strengthen the purpose that natural type of the present invention that the above-mentioned partial sequence by WT1 of mentioning forms is assisted the activity of peptide.Non-peptide the 1st, 4,6 and/or 9 amino acid whose parts of locating to replace can keep natural type sequence (that is, keeping having the partial sequence of WT1), or as long as activity keeping just can further change.

Preferred embodiment comprises 10-25 amino acid whose peptide, it is selected from following amino acid that its 1st, 4,6 and/or 9 of comprising aminoacid sequence as shown in SEQ ID NO:12 locates amino-acid residue: for the 1st, and phenylalanine, tyrosine, tryptophane, α-amino-isovaleric acid, Isoleucine, leucine and methionine(Met); For the 4th, α-amino-isovaleric acid, Isoleucine, leucine, methionine(Met), aspartic acid and L-glutamic acid; For the 6th, l-asparagine, Serine, Threonine, glutamine, Methionin and aspartic acid; And for the 9th, the amino-acid residue of aspartic acid, L-glutamic acid and glutamine replaces, and is combined with HLA-DRB1*0405 and induces the aminoacid sequence of helper T cell.

Still preferred embodiment comprises the 16 derivative amino acid moiety peptides of WT1 as shown in SEQ ID NO:24, it comprises the aminoacid sequence as shown in SEQ ID NO:12, the the 3rd, 6,8 and/or 11 locate amino-acid residue for being selected from following amino acid: for the 3rd, phenylalanine, tryptophane, α-amino-isovaleric acid, Isoleucine, leucine and methionine(Met); For the 6th, α-amino-isovaleric acid, Isoleucine, methionine(Met), aspartic acid and L-glutamic acid; For the 8th, l-asparagine, Serine, Threonine, glutamine, Methionin and aspartic acid; And for the 11st, the peptide that the amino-acid residue of aspartic acid, L-glutamic acid and glutamine replaces.Preferred example can comprise 16-25 amino acid whose peptide, and it comprises the aminoacid sequence from the change of SEQ ID NO:24.

The present invention also provides a kind of the present invention of comprising to assist the peptide (epitope peptide of what is called) of peptide (natural or change peptide) and cancer antigen peptide.

Recently, already reported a kind of epitope peptide, wherein cancer antigen peptide (also referred to as " CTL epi-position ") is connected with auxiliary peptide (also referred to as " auxiliary epi-position "), can effectively induce CTLs.That is to say, helper T cell (CD4 positive T cell) is activated by the auxiliary peptide with various active, described activity comprises induces CTLs differentiation and keeps, and the effector of activation such as scavenger cell etc., therefore is considered to have strengthened CTL-by cancer antigen and induces.Object lesson as the peptide of wherein assisting peptide to be connected with cancer antigen peptide, reported the epitope peptide of the auxiliary peptide composition that the CTLs of effectively anti-various epi-positions in the restricted antigen peptide of HLA-A2-(6 peptide), the restricted antigen peptide of HLA-A11-(3 peptide) and the body in coding HBV-source induces DNA (minigene) (Journal of Immunology 1999,162:3915-3925).In fact, wherein CTL epi-position (corresponding to the tumor antigen peptide of melanoma-associated antigen gp100 280-288 position) has been carried out clinical trial (Clinical Cancer Res. with the peptide that auxiliary peptide (the auxiliary epi-position of T that tetanus toxin is originated) is connected, 2001,7:3012-3024).

Therefore, as specific embodiment, peptide of the present invention also comprises the epitope peptide that comprises the auxiliary peptide of the present invention and cancer antigen peptide.

With regard to cancer antigen peptide, any known cancer antigen peptide all can use; But, preferably use the derivative cancer antigen peptide of WT1 (natural or change peptide).Concrete example comprise restriction HLA-A1 ,-A0201 ,-A0204 ,-A0205 ,-A0206 ,-A0206 ,-A0207 ,-A11 ,-A24 ,-A31 ,-A6801 ,-B7 ,-B8 ,-B2705 ,-B37 ,-Cw0401 ,-the WT1-derived peptide of Cw0602 etc.

The example of the cancer antigen peptide that WT1-is derivative comprise list in WO2000/18795 Table II-Table X LVI those and have peptide as the change of cancer antigen peptide active (in conjunction with HLA antigen and induce the CTLs active).

The cancer antigen peptide that WT1-derives example more specifically comprises as follows:

Cys Met Thr Trp Asn Gln Met Asn Leu (SEQ ID NO：27)

Cys Tyr Thr Trp Asn Gln Met Asn Leu (SEQ ID NO：28)

Arg Met Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO：29)

Arg Tyr Pro Ser Cys Gln Lys Lys Phe (SEQ ID NO：30)

Ser Tyr Thr Trp Asn Gln Met Asn Leu (SEQ ID NO：31)

Ala Tyr Thr Trp Asn Gln Met Asn Leu (SEQ ID NO：32)

Abu Tyr Thr Trp Asn Gln Met Asn Leu (SEQ ID NO：33)

Arg Tyr Thr Trp Asn Gln Met Asn Leu (SEQ ID NO：34)

Lys Tyr Thr Trp Asn Gln Met Asn Leu (SEQ ID NO：35)

Arg Tyr Phe Pro Asn Ala Pro Tyr Leu (SEQ ID NO：36)

Arg Tyr Pro Gly Val Ala Pro Thr Leu (SEQ ID NO：37)

Ala Tyr Leu Pro Ala Val Pro Ser Leu (SEQ ID NO：38)

Asn Tyr Met Asn Leu Gly Ala Thr Leu (SEQ ID NO：39)

Arg Val Pro Gly Val Ala Pro Thr Leu (SEQ ID NO：40)

Arg Tyr Pro Ser Ser Gln Lys Lys Phe (SEQ ID NO：41)

Arg Tyr Pro Ser Ala Gln Lys Lys Phe (SEQ ID NO：42)

Arg Tyr Pro Ser Abu Gln Lys Lys Phe (SEQ ID NO：43)

Ser Leu Gly Glu Gln Gln Tyr Ser Val (SEQ ID NO：44)

Asp Leu Asn Ala Leu Leu Pro Ala Val (SEQ ID NO：45)

Above, " Abu " refers to " Norleucine ".

Wherein, the peptide as shown in SEQ ID NOS:27 and 29 is HLA-A24 antigen and HLA-A2 hla binding peptide, and the peptide as shown in SEQ ID NOS:44 and 45 is the HLA-A2 hla binding peptide.Other peptide is the HLA-A24 hla binding peptide.

One of preferred cancer antigen peptide is as SEQ ID NO:27, shown in 28,29,30,44 or 45.

Epitope peptide of the present invention example more specifically comprises and comprises auxiliary peptide, namely comprise the aminoacid sequence as shown in as arbitrary in SEQ IDNOS:2-23 and have in conjunction with HLA-DRB1*0405 and induce 10-25 the partial peptide that amino acid whose WT1-is derivative of helper T cell activity, and those of the cancer antigen peptide as shown in as arbitrary in above-mentioned SEQ ID NOS:27-45.

Epitope peptide preferably comprise the auxiliary peptide that formed by the aminoacid sequence as shown in SEQ ID NO:24 and as arbitrary in SEQ ID NOS:27-45 as shown in cancer antigen peptide.

More preferably, epitope peptide comprises the auxiliary peptide that is comprised of the aminoacid sequence as shown in SEQ ID NO:24 and as SEQ ID NOS:27-30,44 and 45 cancer antigen peptides shown in arbitrary.

Epitope peptide can be by the synthetic common method preparation of aforementioned peptide.The genetic engineering preparation of the sequence information of polynucleotide that also can be by the epitope peptide that is used for the synthetic common method of DNA and connects based on the multi-epitope wherein of encoding.Specifically, wherein the epitope peptide that connects of multi-epitope can be encoded the polynucleotide of this peptide in known expression vector by insertions, uses the recombinant expression vector transformed host cell that produces, the cultivation transformant, and from culture recovery target peptide and being prepared.These processes can foundation, for example, is described in document (Molecular Cloning, T.Maniatis etc., GSH Laboratory (1983), DNA Cloning, DM.Glover, IRL PRESS (1985)) method in, or described method is carried out hereinafter.

The activity of the epitope peptide of the auxiliary peptide of described conduct can be confirmed according to preceding method.In addition, the activity of described epitope peptide as cancer antigen peptide can be confirmed by the animal pattern that this peptide is given the people, and this animal pattern is described in WO02/47474 or Int J.Cancer.100, and 565-570 is in 2002.

Epitope peptide of the present invention is considered to be used for setting up more effective cancer therapy or prevention, because auxiliary peptide moiety can activate helper T cell (CD4 positive T cell) so that the helper T cell of the activation with various active to be provided in this auxiliary epitope peptide, described activity comprises induces the CTLs differentiation and keeps, and the effector of activation such as scavenger cell, take this further to have strengthened CTL-by cancer antigen peptide and induce.

Can modify the above-mentioned peptide of the present invention (natural, change or epitope peptide) the amino of-terminal amino acid or the carboxyl of C-end amino acid.Wherein N-end and/or the adorned peptide of C-terminal amino acid residue fall into the scope of peptide of the present invention.

The example that can be used for the amido modified group of-terminal amino acid comprises and being selected from: C _1-6The 1-3 kind group of alkyl, phenyl, cycloalkyl and acyl group.Acyl group comprises C _1-6The C of alkyloyl, phenyl substituted _1-6Alkyloyl, C _5-7The carbonyl of cycloalkyl substituted, C _1-6Alkyl sulphonyl, phenyl sulfonyl, C _2-6The carbalkoxy of carbalkoxy, phenyl substituted, C _5-7The carbonyl that cycloalkyloxy replaces, phenyloxycarbonyl etc.

The example of the peptide of modifying at the carboxyl place of C-end amino acid comprises ester class and amides.The ester class specifically comprises C _1-6The C of alkyl ester, phenyl substituted _0-6Alkyl ester, C _5-7Cycloalkyl ester etc.Amides specifically comprises acid amides, is 1 or 2 C _1-6The acid amides that alkyl replaces, be the C of 1 or 2 phenyl substituted _0-6The acid amides that alkyl replaces forms acid amides of 5 to 7 yuan of azacycloalkyls that comprise the amide group nitrogen-atoms etc.

The present invention also provides the polynucleotide of a kind of above-mentioned peptide of the present invention of mentioning of encoding (natural, that change or epitope peptide).The polynucleotide of this code book invention peptide can DNA or the form of RNA exist.Based on the information about the polynucleotide sequence of the aminoacid sequence of peptide of the present invention or its DNA that encodes, polynucleotide of the present invention can easily be prepared.Specifically, use the synthetic common method of DNA or can synthesize by pcr amplification.

The object lesson of polynucleotide comprises those of the above-mentioned epitope peptide of mentioning of coding.More particularly, the example of polynucleotide comprises that coding comprises auxiliary peptide, namely comprise the aminoacid sequence as shown in as arbitrary in SEQ ID NOS:2-23 and have in conjunction with HLA-DRB1*0405 and induce 10-25 the partial peptide that amino acid whose WT1-is derivative of helper T cell activity, and as those of the epitope peptide of the arbitrary described cancer antigen peptide of above-mentioned SEQ ID NOS:27-45.

Preferred example comprises that coding comprises the auxiliary peptide that is comprised of the aminoacid sequence as shown in SEQ ID NO:24 and as the polynucleotide of the epitope peptide of the arbitrary described cancer antigen peptide of SEQ ID NOS:27-45.

Preferred example comprises that coding comprises the auxiliary peptide that is comprised of the aminoacid sequence as shown in SEQ ID NO:24 and as SEQ ID NOS:27-30, the polynucleotide of the epitope peptide of 44 and 45 arbitrary described cancer antigen peptides.

Should " polynucleotide of code book invention peptide " comprise the polynucleotide that can have the activity that is equivalent to peptide of the present invention at the peptide of forbidding under condition with described polynucleotide complementary sequence hybridization and coding.About " hybridizing under condition forbidding ", " hybridization " can be according to being described in, for example as used herein, Sambrook J., Frisch E.F., Maniatis T. compiles, Molecular Cloning the 2nd edition, Cold Spring Harbor Laboratory press, ordinary method carry out.Term " forbid condition " refers to that hybridization is containing 6xSSC (10 x SSC=1.5M NaCl, 1.5M trisodium citrate), carry out under 45 ℃ in the solution of 50% methane amide, then wash (Molecular Biology, John Wiley﹠amp in 2xSSC solution under 50 ℃; Sons, N.Y. (1989), 6.3.1-6.3.6)) etc.

Can be integrated into by the polynucleotide with above-mentioned preparation in expression vector to build the recombinant expression vector that is used for expressing peptide of the present invention.

Comprise plasmid, phage vector, virus vector etc. at this available expression vector.

When the host was intestinal bacteria (Escherichia coli), the example of carrier comprised such as pUC118, pUC119, pBR332, the plasmid vector of pCR3 etc.; And such as λ ZAPII, the phage vector of λ gt11 etc.When the host was yeast, the example of carrier comprised pYES2, pYEUra3 etc.When the host was insect cell, the example of carrier comprised pAcSGHisNT-A etc.When the host was zooblast, the example of carrier comprised such as pKCR, pCDM8, pGL2, pcDNA3.1, pRc/RSV, the plasmid vector of pRc/CMV etc.; And such as the virus vector of retroviral vector, adenovirus carrier, adeno-associated virus vector etc.

The factor of the marker gene that expression vector optionally contains all if gene of the promotor of abduction delivering, coded signal sequence, be used for selecting, terminator etc.

In addition, expression vector can contain allow peptide with Trx, Histidine (His) label, GST (glutathione S-transferase) etc. as expressing fusion protein in order to promote to separate appended sequence with purifying.Available carrier comprises the gst fusion protein carrier that contains the suitable promotor (lac, tac, trc, trp, CMV, SV40 early promoter etc.) that works in host cell, for example pGEX4T under these circumstances; The carrier that contains Tag sequence (Myc, His etc.), for example pcDNA3.1/Myc-His; And the carrier that can express Trx and histidine-tagged fusion rotein, for example pET32a.

Can be by contain the transformant of carrier of the present invention with the above-mentioned expression vector transformed host cell preparation that obtains.

Comprise intestinal bacteria, yeast, insect cell and zooblast at this available host cell.Colibacillary example comprises such as HB101, C600, JM109, the e. coli k-12 bacterial strain of DH5 α and AD494 (DE3).The example of yeast comprises yeast saccharomyces cerevisiae (Saccharomycescerevisiae).The example of zooblast comprises L929, BALB/c3T3, C127, CHO, COS, Vero and Hela cell.The example of insect cell comprises sf9.

Can use the ordinary method that is suitable for above-mentioned various host cells that expression vector is imported in host cell.Specifically, can use calcium phosphate method, DEAE-dextran method, electroporation and use to be used for liposome method (Lipofectamine, the Lipofectin of transgenosis; Gibco-BRL) import.After importing, culturing cell in containing the conventional medium of selectable marker, the transformant that takes this to contain expression vector can be selected.

Peptide of the present invention can produce by cultivating transformant in (under the condition that this Toplink is expressed) under suitable condition.But the biochemical purification program of the peptide establishing criteria that produces is further separated and purifying.This purifying procedure comprise saltout, ion exchange chromatography, adsorption chromatography, affinity chromatography, gel permeation chromatography etc.As mentioned above, as with the expressing fusion protein of Trx, histidine-tagged, GST etc. the time, this peptide can separate by suitable purifying procedure and purifying when polypeptide of the present invention, and this program has been utilized the characteristic of fusion rotein or label.

The invention provides the antibody of a kind of specific binding peptide of the present invention.This antibody of the present invention is not limited to any form, can be polyclone or the monoclonal antibody of anti-peptide of the present invention as antigen.

As mentioned above, under the condition of its specific binding peptide of the present invention, do not limit antibody of the present invention.The example of preferred antibody comprises that specific binding assists peptide, namely comprise the aminoacid sequence as shown in as arbitrary in SEQ IDNOS:2-23 and have in conjunction with HLA-DRB1*0405 and induce 10-25 the partial peptide that amino acid whose WT1-is derivative of helper T cell activity, those.The more preferably antibody of the auxiliary peptide that formed by the aminoacid sequence as shown in SEQ ID NO:24 of specific binding.

The method of Dispersal risk is well-known in this area, and antibody of the present invention can be prepared according to arbitrary ordinary method (Current protocols in Molecular Biology edits Ausubel etc. the .John Wiley﹠amp of (1987) publisher; The Sons.11.12-11.13 joint, Antibodies; A Laboratory Manual, Lane, H, the volumes such as D., Cold SpringHarber Laboratory Press, New York 1989).

Specifically, but the peptide of antibody the application of the invention of the present invention and reclaims antibody to obtain as the non-human animal of antigen immune such as rabbit from this immune animal serum with ordinary method.With regard to monoclonal antibody, they can be by the non-human animal with peptide immunity of the present invention such as mouse, the splenocyte and the myeloma cell that produce are carried out cytogamy, and reclaim from the hybridoma that produces monoclonal antibody with obtain (Current protocols inMolecular Biology edits Ausubel etc. the .John Wiley﹠amp of (1987) publisher; The Sons.11.4-11.11 joint).

When depending on that the host uses different adjuvants to strengthen immunne response, the antibody of anti-peptide of the present invention also can produce.The example of adjuvant comprises Fu Shi (Freund) adjuvant; Mineral coagulant such as aluminium hydroxide; Such as lysolecithin,

The tensio-active agent of polyvalent alcohol, polyanion, peptide, oil-emulsion, key hole keyhole limpet hemocyanin and dinitrophenol(DNP); Such as BCG (Bacille deCalmette-Guerin) or coryneform people's adjuvant etc.

As mentioned above, the antibody of identification peptide of the present invention and its active antibody of neutralization can easily be prepared by immune animal in a usual manner.Antibody can be used for affinity chromatography, immune diagnostic method etc.Immune diagnostic method can suitably be selected from immunoblotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), fluorescence or luminescence assays etc.This immune diagnostic method is effective in the cancer diagnosis such as the expression WT1 gene of cancer of the stomach, colorectal carcinoma, lung cancer, mammary cancer, protoblast cancer, skin carcinoma, bladder cancer, prostate cancer, uterus carcinoma, cervical cancer, ovarian cancer etc.

The invention provides a kind of pharmaceutical composition, comprise peptide of the present invention (natural, change with the epitype peptide), contain the expression vector of polynucleotide of the present invention or contain the cell of expression vector of the present invention, and pharmaceutically acceptable carrier.This pharmaceutical composition can be used as the inductor of helper T cell or the toughener of cancer vaccine effect, the following detailed description in detail effectively.

(1) a kind of helper T cell inductor (toughener of cancer vaccine effect) that comprises as the peptide of the present invention of effective constituent

Peptide of the present invention has the activity of inducing helper T cell, and the helper T cell of the inducing activity of again can enhanced CT L-inducing, and it acts on cancer vaccine by the differentiation of inducing CTLs and the effector of keeping and activating such as scavenger cell.Therefore, the invention provides a kind of toughener (as the pharmaceutical composition that is used for strengthening cancer vaccine effect reagent) that comprises as the cancer vaccine effect of the peptide of the present invention of effective constituent.When toughener of the present invention gave the patient of the HLA-DRB1*0405-positive and the WT1-positive, this peptide was the HLA-DRB1*0405 antigen that is handed to antigen presenting cell; Induce and activate the specificity helper T cell (CD4 positive T cell) of this peptide of identification and HLA-DRB1*0405 antigenic compound; The helper T cell of this activation can be brought into play to relate to and induce CTLs differentiation and keep and activate activity such as the effector of scavenger cell.In this mode, be enhanced as the activity of the activation of cancer vaccine effect and the CTLs that induces.

Cancer vaccine effect toughener of the present invention can be used for preventing or treats cancer with the WT1 gene expression dose that raises, for example, such as the leukemia of leukemia, the abnormal syndrome of spinal cord development, multiple myeloma and malignant lymphoma, and such as the solid carcinoma of cancer of the stomach, colorectal carcinoma, lung cancer, mammary cancer, protoblast cancer, liver cancer, skin carcinoma, bladder cancer, prostate cancer, uterus carcinoma, cervical cancer and ovarian cancer.

Cancer vaccine effect toughener of the present invention can give before cancer vaccine gives or after giving simultaneously.

This comprises the cancer vaccine effect toughener that gets peptide of the present invention as effective constituent can contain auxiliary peptide or epitope peptide as effective constituent, and wherein said peptide is connected with cancer antigen peptide (CTL epi-position).As mentioned above, shown that recently cancer antigen peptide in epitope peptide (CTL epi-position) and auxiliary peptide (auxiliary epi-position) can induce CTLs effectively.When giving the epitope peptide of this form, described peptide inserts in antigen presenting cell; In the antigen peptide that produces of degrading in born of the same parents, auxiliary peptide and MHC II class antigen (HLA-DRB1*0405) combination, and cancer antigen peptide is combined with MHC I class antigen; Therefore formed corresponding mixture is with high-density and is handed to the antigen presenting cell surface.When helper T cell identification HLA-DRB1*0405 antigen and auxiliary peptide complex, the activity that CTL-induces, namely the effect of cancer vaccine, be further enhanced as the effector result of inducing the CTLs differentiation and keep and activate such as scavenger cell.On the other hand, when CTLs identifies the mixture of cancer antigen peptide and MHC I class antigen, CTLs propagation and destruction of cancer cells.Therefore, the pharmaceutical composition of the present invention itself that comprises as the epitope peptide of the present invention of effective constituent can be used as cancer vaccine, and the toughener of cancer vaccine effect.

This comprise as the cancer vaccine effect toughener of the peptide of the present invention of effective constituent can with pharmaceutically acceptable carrier, for example, suitable adjuvant gives together, or makes the cellular immunization performance effectively set up with the form of particle.About adjuvant, those are described in document (Clin.Microbiol.Rev., 7:277-289,1994) etc. is available.Concrete example comprise composition, the marine organisms source of microbe-derived composition, cytokine, plant origin composition, such as the mineral coagulant of aluminium hydroxide, such as lysolecithin and

The tensio-active agent of polyvalent alcohol, polyanion, peptide, oil-emulsion (emulsion preparations) etc.Also comprise Liposomal formulation, wherein composition is bonded to microparticle formulation on the pearl with several micron diameters, wherein composition invests the preparation of lipid etc.

Can for example carry out intracutaneous, subcutaneous, intramuscular or intravenous administration.Although the dosage of peptide of the present invention can be depending in preparation, the disease that for example will treat, patient's age and body weight are suitably regulated, its usually in the 0.0001mg-1000mg scope, preferred 0.001mg-1000mg, more preferably 0.1mg-10mg, gave once in preferred every several days to every several months.

The present invention also provides a kind of pharmaceutical composition that comprises peptide of the present invention and cancer antigen peptide combination.According to Combination application of the present invention, strengthened by peptide of the present invention as the cancer antigen peptide effect (namely inducing and activate the CTLs activity) of cancer vaccine, and can more effectively be carried out treatment and the prevention of cancer.

Term " combination " comprises two kinds of forms, and namely peptide of the present invention and cancer antigen peptide give with mixed form or unpack format.

Can use the preparation as the peptide of mixture of containing of previous preparation, or the preparation that comprises respectively corresponding peptides that will before prepare before use makes up to carry out the giving of peptide of mixed form.

In the situation that peptide gives with unpack format, this separation preparation can give in the timed interval successively, or gives simultaneously.When carrying out administration with the timed interval, peptide of the present invention (cancer vaccine effect toughener) and cancer antigen peptide (cancer vaccine) can sequentially give or give in reverse order according to this.

The example of the combination of peptide of the present invention and cancer antigen peptide comprises test kit.

With regard to the cancer antigen peptide that can be used in combination with peptide of the present invention, any cancer antigen peptide known to usually can use, and example comprises the cancer antigen peptide (natural type, change type) that WT1-is derivative.Concrete example comprises the cancer antigen peptide as shown in as arbitrary in SEQ ID NOS:27-45, is preferably SEQ ID NOS:27-30,44 and 45 cancer antigen peptides shown in arbitrary.

(2) comprise helper T cell inductor (toughener of cancer vaccine effect) as the expression vector of the present invention of effective constituent

Contain coding peptide of the present invention, be similar to the expression vector of polynucleotide of the peptide of the invention described above, have the activity of inducing helper T cell, and can be used as the effective constituent of cancer vaccine effect toughener of the present invention.Therefore, the invention provides a kind of expression vector of the polynucleotide that contain the peptide of the present invention of encoding that comprises as the cancer vaccine effect toughener (namely as the pharmaceutical composition that strengthens cancer vaccine effect reagent) of effective constituent.

Recently, code displaying wherein the polynucleotide of the epitope peptide that is connected of cancer antigen peptide (CTL epi-position) and auxiliary peptide (auxiliary epi-position) have the activity of effectively inducing CTLs in body.For example, reported the restricted antigen peptide of HLA-A2-(6 peptide), the restricted antigen peptide of HLA-A11-(3 peptide) in the HBV-wherein source of encoding and be connected in DNA (minigene) body of the epitope peptide that peptide connects and induce effective CTLs (Journal of Immunology1999,162:3915-3925) for various epi-positions.

The polynucleotide of the epitope peptide that therefore, the effective constituent of cancer vaccine effect toughener can be by the invention described above of encoding are integrated in suitable expression vector and obtain.

When the expression vector that contains as the polynucleotide of the present invention of cancer vaccine effect toughener effective constituent, following method can be used.

With regard to regard to the method for the expression vector transfered cell that will contain polynucleotide of the present invention, comprise that any method or the additive method that those utilize virus vector can use (Nikkei-Science, in April, 1994, p20-45; Gekkan-Yakuji, 36 (1), p23-48 (1994); Jikken-Igaku-Zokan, 12 (15), 1994, and the document of wherein quoting as proof).

Utilize the example of virus vector method to comprise DNA or the RNA viruses that wherein DNA of the present invention is integrated into such as retrovirus, adenovirus, adeno associated virus, simplexvirus, vaccinia virus, poxvirus, poliovirus or Syndebis (Sindbis) virus, then those in transfered cell.Especially, it is particularly preferred utilizing the method for retrovirus, adenovirus, adeno associated virus or vaccinia virus etc.

The example of additive method comprises the wherein direct intramuscularly of expression vector (DNA inoculation), liposome method, fat transfection (Lipofectin) method, microinjection, calcium phosphate method and electroporation.DNA inoculation and liposome method are particularly preferred.

As in practice with the method for expression vector of the present invention as medicinal application, exist expression vector wherein directly import in body in body method and wherein expression vector in some isolated cells of external importing, then cell is imported again method (Nikkei-Science in the external rear body of elder generation in body, in April, 1994,20-45; Gekkan-Yakuji, 36 (1), 23-48 (1994); Jikken-Igaku-Zokan, 12 (15), 1994, and the document of wherein quoting as proof).In body, method is preferred.

With regard to method in body, depend on disease and the symptom that will treat, can complete administration by any suitable approach.For example, can pass through the administrations such as intravenously, intra-arterial, subcutaneous, intracutaneous, intramuscular approach.When method was carried out administration in by body, composition can carry out administration by various forms, and for example solution, particularly preparation, for example, to contain the injection liquid form as the expression vector of the present invention of effective constituent, if necessary, also can add conventional carrier.As for the liposome (for example Sendai virus (HVJ)-liposome) that the liposome that contains expression vector of the present invention or film merge, they can exist such as the Liposomal formulation form of the freezing medicine of suspension, freezing medicine, centrifugal concentrating etc.

Although depend on, for example, the disease that treat, patient's age and body weight, in preparation, the content of expression vector can suitably be regulated, but common 0.0001mg-100mg, the expression vector of the present invention of preferred 0.001mg-10mg can give once in every several days to every several months.

When the expression vector of the invention described above gave the HLA-DRB1*0405-positive and WT1-positive patient, peptide of the present invention was the HLA-DRB1*0405 antigen that is handed to antigen presenting cell; Induced and activated the specificity helper T cell (CD4 positive T cell) of identifying this peptide and HLA-DRB1*0405 antigenic compound; The helper T cell of this activation can be brought into play to relate to and induce CTLs differentiation and keep and activate activity such as the effector of scavenger cell.In this mode, be enhanced as the activity of inducing CTLs of cancer vaccine effect.Comprising cancer vaccine effect toughener as the expression vector that contains polynucleotide of the present invention of effective constituent can be used for preventing or treats cancer with the WT1 gene expression dose that raises, for example, such as the leukemia of leukemia, the abnormal syndrome of spinal cord development, multiple myeloma and malignant lymphoma, and such as the solid carcinoma of cancer of the stomach, colorectal carcinoma, lung cancer, mammary cancer, protoblast cancer, liver cancer, skin carcinoma, bladder cancer, prostate cancer, uterus carcinoma, cervical cancer and ovarian cancer.

In the expression vector situation of the above-mentioned polynucleotide that contain the epitope peptide of encoding, antigen presenting cell has been integrated this expression vector and has been produced antigen peptide by degraded in born of the same parents, wherein auxiliary peptide and cancer antigen peptide are combined with MHC II class antigen (HLA-DRB1*0405) and MHC I class antigen respectively, form mixture.Formed mixture is with high-density and is handed to the antigen presenting cell surface.When helper T cell identification HLA-DRB1*0405 antigen and auxiliary peptide complex, the CTL-induced activity, namely the cancer vaccine effect, be further enhanced by the effector of inducing the CTLs differentiation and keep and activate such as scavenger cell.On the other hand, when CTLs identification cancer antigen peptide and MHC I class antigenic compound, CTLs propagation and destruction of cancer cells.Therefore, the pharmaceutical composition of the present invention itself that comprises as the expression vector of the polynucleotide that contain code book invention epitope peptide of effective constituent can be used as cancer vaccine, and the toughener of cancer vaccine effect.

It is a kind of by 10-25 peptide that continuous amino acid forms in the people WT1 aminoacid sequence as shown in SEQ ID NO:1 that the present invention also provides, and described peptide is in conjunction with HLA-DRB*1502 and induce helper T cell.The present invention comprise the N-end of amino-acid residue wherein and/or the C-end adorned in conjunction with the antigen peptide of HLA-DRB1*1502 or wherein indivedual amino-acid residues reformed those.

Can be similar to the method for above-mentioned antigen peptide of the present invention in conjunction with HLA-DRB1*0405 synthetic in conjunction with HLA-DRB1*1502 antigen peptide and measure its activity.

HLA-DRB1*1502-conjugated antigen peptide of the present invention is a kind of by 10-25 partial peptide that continuous amino acid forms in the people WT1 aminoacid sequence as shown in SEQ ID NO:1.Preferred peptide is that those are comprised of 13-17 continuous amino acid in people WT1 aminoacid sequence.

The present invention is based on finder WT1 and contains and have in conjunction with HLA-DRB1*1502 and induce the antigen peptide part of helper T cell activity.Use the software (Propred that is used for prediction MHC II class binding peptide, Bioinformatics 17:1236,2001) carry out the search of the nine amino acid part (the nine amino acid part that can be suitable for MHC II quasi-molecule peptide binding groove groove) that may be combined with HLA-DRB1*1502.The example of the nine amino acid part of the WT1 that identifies is shown in SEQ ID NOS:46-56.Therefore, the object lesson of HLA-DRB1*1502 conjugated antigen peptide of the present invention comprises and comprises the aminoacid sequence as shown in as arbitrary in SEQ ID NOS:46-56, in conjunction with HLA-DRB1*1502 and induce the peptide of helper T cell.

Described peptide preferably is comprised of about 10-25 amino acid, more preferably is comprised of about 13-17 amino acid.The example of preferred embodiment comprises the partial peptide that is comprised of 10-25 amino acid (preferably by 13-17 amino acid) that WT1 is derivative, it comprises the aminoacid sequence as shown in SEQ ID NO:50, and has in conjunction with HLA-DRB1*1502 and the activity of inducing helper T cell.Still the example of preferred embodiment comprises the partial peptide that is comprised of 16-25 amino acid (preferably by 16-17 amino acid) that WT1 is derivative, it comprises the aminoacid sequence as shown in SEQ IDNO:24, and has in conjunction with HLA-DRB1*1502 and the activity of inducing helper T cell.Aminoacid sequence as shown in SEQ ID NO:24 represents the 16 amino acid whose partial peptides that WT1 is derivative, and comprises the aminoacid sequence as shown in SEQ ID NO:50.

The WT1 that preferred embodiment is comprised of the aminoacid sequence as shown in SEQ ID NO:24 _332-347Peptide.

Described WT1 _332-347Peptide is that a kind of non-kind is the auxiliary peptide of selectivity, and it not only is combined with the HLA-DRB1*0405 molecule, also is combined with the HLA-DRB1*1502 molecule.Therefore, WT1 _332-347Be a kind of auxiliary peptide of having HLA-DRB1*0405 patient and having equally HLA-DRB1*1502 patient of can be applicable to, therefore the viewpoint from patient's broad field of application is useful.

Epitope peptide, polynucleotide, antibody and the pharmaceutical composition that HLA-DRB1*1502-conjugated antigen peptide of the present invention is relevant can be similar to the method preparation of the invention described above HLA-DRB1*0405-conjugated antigen peptide and use.

Embodiment

The present invention further illustrates by following embodiment, is subject to this but should not be construed as the present invention.

Embodiment 1

1. preparation dendritic cell

Use Ficoll-Paque (Ficoll-Paque) separating periphery blood monocytic cell (PBMCs) from the positive healthy volunteer's blood of HLA-DRB1*0405-by density gradient centrifugation.With resulting 8 * 10 ⁶Individual PBMCs is suspended in the 2mlX-VIVO 15 that contains 1%AB serum ^TMIn substratum (Camblex), be seeded in 6 hole culture plates, cultivated 2 hours.After cultivation, remove not attached cell, with Hanks solution washing attached cell.At the X-VIVO15 that contains 1%AB serum, 1000U/ml IL-4 and 1000U/ml GM-CSF ^TMCultivate attached cell in substratum.At the 2nd and 4 day that cultivates, half substratum is replaced into fresh culture.At the 6th day, it was 100U/ml that interpolation TNF-α makes its final concentration.Cell survival in the 7th day is used as the test dendritic cell.

2. prepare CD4 positive T cell (helper T cell)

Described in above-mentioned (1), use the blood that obtains from same healthy volunteer.With the RPMI substratum with 2 times of hemodilutions.Add the blood of about 100ml dilution to mixtures of antibodies (cocktail) for separating of the CD4 positive T cell, RosetteSep ^TM(Stemcell) in, and with mixture at room temperature standing 20 minutes.Then, use Ficoll-Paque (Ficoll-Paque) to collect the CD4 positive T cell by density gradient centrifugation.

3. induce WT1 peptide specific CD4 positive T cell

Use predictor (Propred, Bioinformatics 17:1236,2001) at aminoacid sequence (ncbi database, the accession number P19544 of WT1 albumen, XP_034418, SEQID NO:1) middle search may be in conjunction with the peptide of HLA-DRB1*0405.Select and synthesized 3 kinds of peptides.These peptides have with those and are present in the identical aminoacid sequence of the aminoacid sequence of WT1 following position:

172-186 position: PNHSFKHEDPMGQQG (WT1 _172-186, SEQ ID NO:25);

225-243 position: NLYQMTSQLECMTWNQMNL (WT1 _225-243, SEQID NO:26); And

332-347 position: KRYFKLSHLQMHSRKH (WT1 _332-347, SEQ IDNO:24).

With 3 * 10 ⁵Cells/well is seeded in the dendritic cell of preparation in above-mentioned (1) in 24 hole culture plates, and adds peptide to the 50 μ g/ml of SEQ ID NO:24.After cultivating 4 hours, (25Gy) stops Growth of Cells by x-ray irradiation.With 3 * 10 ⁶Cells/well is added the CD4 positive cell of preparation in above-mentioned (2) in each hole to, and cultivates altogether with dendritic cell.As for substratum, use the X-VIVO 15 that contains 1%AB serum ^TMSubstratum.After cultivating beginning, replaced the substratum of half and added IL-2 to 20U/ml with fresh culture in every 2 days.From cultivating the 7th and 14 day of beginning, collect the T cell and with 3 * 10 ⁶Cells/well is seeded in 24 hole culture plates, adds in the hole more in addition and has used 20 μ g/ml peptides (SEQ ID NO:24) to stimulate and stood 3 * 10 of x-ray irradiation (25Gy) ⁵Dendritic cell.Follow co-cultured cell.As for substratum, use the X-VIVO 15 that contains 1%AB serum and 20U/ml IL-2 ^TMSubstratum.

After stimulating for the 3rd time, reclaim the T cell and with 3 * 10 ⁴Cells/well is seeded in 96 hole flat boards.With 3 * 10 ⁴The cells/well interpolation has used 20 μ g/ml peptides (SEQ ID NO:24) to stimulate and stand the dendritic cell of x-ray irradiation (25Gy), then carries out common cultivation.As negative control group, the T cell is cultivated altogether with the dendritic cell that do not stimulate with peptide, as positive controls, adds 0.2%PHA and replaces dendritic cell.After cultivating 80 hours, add [ ³H]-thymidine (37kBq/ hole), cell was cultivated 16 hours again.Then, use β-scintillometer measurement mix in cell [ ³H]-thymidine.The results are shown in Fig. 1.When with use WT1 _332-347When the dendritic cell that stimulate are cultivated altogether, with the peptide (WT1 of WT1 332-347 position _332-347, SEQ ID NO:24) and the CD4 positive T cell that stimulates shown that propagation replys.But when cultivating altogether with the dendritic cell that do not stimulate with peptide, or when cultivating altogether with the dendritic cell that peptide with the aminoacid sequence with the SEQID NO:25 that is different from SEQ ID NO:24 or 26 stimulates, the CD4 positive T cell does not show that propagation replys.These results show WT1 _332-347Peptide (SEQ IDNO:24) has been induced specific C D4 positive T cell as antigen peptide.

Embodiment 2

Foundation is specific to the CD4 positive T cell system of WT1 peptide

The dendritic cell that will prepare with the method that is similar to embodiment 1 are with 10 ⁴Cells/well is seeded in 96 hole flat boards, then with 10 ³The cells/well inoculation is WT1 _332-347The CD4 positive T cell that peptide (SEQ ID NO:24) is induced.As for substratum, use the X-VIVO 15 that contains 1%AB serum, 20U/mlIL-2 and 5 μ g/ml PHA ^TMSubstratum.Set up CD-4 positive T cell system and called after " G2 clone " by cultured continuously.Measure G2 clone replying the dendritic cell that stimulate with peptide by the method that is similar to claim 1.The results are shown in Fig. 2.When with use WT1 _332-347When the dendritic cell that peptide stimulates were cultivated altogether, G2 clone has shown to breed replied, and when cultivating altogether with the dendritic cell that do not carry out the peptide stimulation, did not show to breed and replied.

These results show that G2 clone is a kind of WT1 of being specific to _332-347The CD4 positive T cell system of peptide.

Embodiment 3

The antigen presentation of WT1 peptide is to the HLA-DR molecule

To be similar to the method for embodiment 1, use Ficoll-Paque (Ficoll-Paque) separating periphery blood monocytic cell (PBMCs) from the positive healthy volunteer's blood of HLA-DRB1*0405-by density gradient centrifugation.Then, with 10 ⁷Cells/well is seeded in PBMCs in 24 hole flat boards.As for substratum, use the RPMI1640 substratum that contains 10%FCS and 55 μ M 2ME.After interpolation contains the substratum of Epstein-Barr virus (EBV), then 4 weeks of cultured continuously are to set up the B-clone that transforms with EBV, this clone called after " B-LCL (-) cell ".The EBV preparation is in the culture supernatants of B95-8 (JCRB Cell Bank No.9123), and this B95-8 is the clone of a kind of EBV of generation.With B-LCL (-) Cell regulate to 3 * 10 ⁷Cell/mL, and add wherein and contain the substratum of expressing the WT1 gene viruses then adds polypropylene (final concentration, 8 μ g/mL), and adds in the flat board of this mixture to 24 hole with the 1ml/ hole.Cultivate after 16 hours, add the 1ml fresh culture in each hole, and proceed to cultivate.Add G418 (Liu Suanyan NEOMYCIN SULPHATE) in each hole to 0.7 μ g/mL, when the cell of quiding gene is selected, culture plate 5-7 days.B-clone called after " B-LCL (+) cell " with selected expression WT1.According to Blood, 89:1405, the method for describing in 1997, the amount of the WT1 gene by RT-PCR technical measurement B-LCL (-) and B-LCL (+) cell expressing.This measuring result is changed by being assumed to 1 as the expression amount of the K562 clone of positive control.The value of resulting B-LCL (-) cell is 1.6 * 10 ^-4, and the value of B-LCL (+) cell is 3.2, shows that the WT1 gene is high expression level.To be similar to method research G2 cell the replying B-LCL (+) cell of embodiment 2.In test group, with the G2 cytomixis before, restricted to confirm HLA-DR-with anti-HLA-DR antibody treatment B-LCL (+) cell.The results are shown in Fig. 3.It has disclosed when when expressing B-LCL (+) co-culture of cells of endogenous WT1 gene, is that G2 has shown that propagation replys to the CD4 positive T cell of the peptide positive, and described replying suppressed by anti--HLA-DR antibody.These results show WT1 _332-347Peptide is to produce in WT1 albumen born of the same parents, and is as the antigen endogenous and is handed to the HLA-DR molecule.

Embodiment 4

Foundation is specific to WT1 _332-347 The CD4 positive T cell of peptide is E04.1

Except the TNF-α final concentration interpolation in the 6th day is 200IU/ml, use the blood that separates from the positive healthy volunteer of HLA-DRB1*0405-to prepare dendritic cell with the method that is similar to embodiment 1.Use the blood that obtains from the same healthy volunteer who is used for the preparation dendritic cell to prepare the CD4 positive T cell.Specification sheets separation of C D4 positive T cell according to the RosetteSep (StemCell) that is used for the separation of CD4 positive T cell.

To be similar to the method for embodiment 1, above-mentioned dendritic cell and CD4 positive T cell are used for inducing being specific to WT1 peptide (SEQ ID NO:24, WT1 _332-347) the CD4 positive T cell.Be specific to WT1 by what the limiting dilution technique cultured continuously produced _332-347The CD4 positive T cell of peptide is E04.1 to set up the CD4 positive T cell.As the feeder cell in this limiting dilution finishes, the PBMCs that processes with the method preparation that is similar to embodiment 1 and by x-ray irradiation is with 1 * 10 ⁵Cells/well is inoculated.As for substratum, use the X-VIVO 15 that contains 20IU/ml IL-2 and 5 μ g/ml PHA ^TMSubstratum.

Except add [ ³H]-cultivate after thymidine and continue to carry out outside 18 hours, measure E04.1 clone to using WT1 by the method that is similar to embodiment 1 _332-347Replying of the dendritic cell that peptide stimulates.The results are shown in Fig. 4.When with use WT1 _332-347When the dendritic cell that peptide stimulates were cultivated altogether, the E04.1 cell showed that propagation replys, and when cultivating altogether with the dendritic cell that do not stimulate with this peptide, did not show to breed and reply.These results show that the E04.1 cell is a kind of WT1 of being specific to _332-347The CD4 positive T cell system of peptide.

Embodiment 5

WT1 _332-347 The specific binding of peptide and HLA-DR

With the E04.1 cell set up in embodiment 4 with 1 * 10 ⁴Cells/well is seeded in 96 hole culture plates.B clone B-LCL (-) cell of the positive healthy volunteer's blood of foundation HLA-DRB1*0405-in embodiment 3 is with the WT1 of 20 μ g/ml concentration _332-347Peptide stimulates, and processes by x-ray irradiation, with 3 * 10 ⁴Cells/well is seeded in 96 hole flat boards, and with the E04.1 co-culture of cells.As negative control group, B-LCL (-) cell and the E04.1 co-culture of cells that will stimulate without peptide.

As test group, with 20 μ g/ml anti--HLA-DR antibody (G46.6, BDParMingen), anti--HLA-I antibody-like (G46-2.6, BD ParMingen) or anti--HLA-DQ antibody (SPVL3, Immunotech) processes and used WT1 _332-347Peptide stimulates and stands B-LCL (-) cell 30 minutes of x-ray irradiation, and restricted to confirm HLA-DR with the E04.1 co-culture of cells.As the negative control group of antibody treatment, the cell that anti--mouse IgG antibody is processed in a similar fashion with the E04.1 co-culture of cells.

After cultivating altogether, measure the growth of E04.1 cell with the method that is similar to embodiment 4.The results are shown in Fig. 5.When with use WT1 _332-347During B-LCL (-) co-culture of cells that peptide stimulates, the E04.1 cell has shown that propagation replys.But, when using anti--HLA-DR antibody treatment WT1 _332-347During B-LCL (-) cell that peptide stimulates, Growth of Cells is suppressed.In addition, for the WT1 with other antibody treatment _332-347B-LCL (-) cell that peptide stimulates, E04.1 cell have shown to breed replys, but B-LCL (-) cells that stimulates without peptide is not shown to breed replys.These results show WT1 _332-347Peptide specific is in conjunction with the HLA-DR in the HLA molecule, and induces and be specific to WT1 _332-347The growth of the positive E04.1 clone of the CD4 of peptide.

Embodiment 6

WT1 _332-347 The specific binding of peptide and HLA-DRB1*0405

Prepare PBMCs with the method that is similar to embodiment 4 from HLA-DRB1*0405-positive or negative healthy volunteer blood.Then, PBMCs is with the WT1 of 20 μ g/ml _332-347Peptide stimulates and stands x-ray irradiation, with 3 * 10 ⁴Cells/well is seeded in 96 hole flat boards.Then, with 1 * 10 ⁴Cells/well is inoculated into the E04.1 cell in this 96 hole flat board, co-cultured cell.As negative control group, PBMCs and the E04.1 co-culture of cells that will stimulate without peptide.

After cultivating altogether, measure the growth of E04.1 cell with the method that is similar to embodiment 4.The results are shown in Fig. 6.Donor 1 (HLA-DRB1*0405/0803) and donor 2 (HLA-DRB1*0405/0101) are the HLA-DRB1*0405-positives, cultivate altogether and use WT1 with the PBMCs that separates from each donor _332-347The E04.1 cell that peptide stimulates has shown to breed replys.On the other hand, donor 3 (HLA-DRB1*0101/1001) and donor 4 (HLA-DRB1*1201/0802) are the HLA-DRB1*0405-feminine genders, cultivate altogether and use WT1 with the PBMCs that separates from each donor _332-347The E04.1 cell that peptide stimulates does not show to breed replys.In addition, in all cases, when using the PBMCs that stimulates without peptide, do not observe propagation and reply.The above results shows WT1 _332-347HLA-DRB1*0405 specific binding in the HLA-DRB1 molecule of peptide and demonstration polymorphism, and induce WT1 _332-347The growth of specific C D4 positive cell line E04.1.

Embodiment 7

WT1 _332-347 The peptide antigen presentation is to HLA-DRB1*0405

As described in Example 3, set up B-LCL (-) cell (B-clone) and B-LCL (+) cell (the B-clone of expression WT1) from the positive healthy volunteer's blood of HLA-DRB1*0405-, stand respectively x-ray irradiation and with 3 * 10 ⁴Cells/well is seeded in 96 hole flat boards.The E04.1 cell is with 1 * 10 ⁴Cultivation is inoculated and be total to cells/well.Then the growth of measuring the E04.1 cell with the method that is similar to embodiment 4 is replied.The results are shown in Fig. 7.When expressing B-LCL (+) co-culture of cells of WT1, the E04.1 cell has shown that propagation replys, but when not expressing B-LCL (-) co-culture of cells of WT1, does not show that propagation replys.

Then, apoptosis-induced B-LCL (-) or B-LCL (+) cell (are respectively 1 * 10 ⁵Cell) with the dendritic cell (3 * 10 that prepare with the method that is similar to embodiment 4 from the positive healthy volunteer's blood of HLA-DRB1*0405- ⁴Cell) cultivate altogether 16 hours, be seeded in the hole of 96 hole flat boards, and with E04.1 cell (1 * 10 ⁴Cell) cultivate altogether.Then, the growth of measuring the E04.1 cell with the method that is similar to embodiment 1 is replied.The results are shown in Fig. 8.When cultivating altogether with the dendritic cell of B-LCL (+) cytositimulation of apoptosis induction, the E04.1 cell has shown that propagation replys, but when cultivating altogether with the dendritic cell of B-LCL (-) cytositimulation of apoptosis induction, does not show to breed and reply.These results show WT1 _332-347At first peptide is to produce by the WT1 albumen of degrading in B-LCL (+) cell, then is the propagation that is handed to HLA-DRB1*0405 and induces the E04.1 cell.

Carry out the inducing of apoptosis of B-LCL (-) and B-LCL (+) cell by osmotic shock.That is, with 1 * 10 ⁶Cell suspension in the height of 500 μ l oozes substratum (containing 0.5M sucrose, 10%w/v cetomacrogol 1000 and 10mM HEPES, the RPMI substratum of pH 7.2), and under 37 ℃ standing 10 minutes.Then before being adjusted to 37 ℃, dilute 30 times of this cultures with hypotonic substratum (60%RPMI, 40% water), and under 37 ℃ standing 2-3 minute.By at room temperature centrifugal 5 minutes collecting cells and be used as the apoptosis induction cell.Prove conclusively inducing of apoptosis by the fluorescence dye (iodate the third ingot, and annexin V, i.e. phosphatidylserine-binding reagents) that is used for dead cell dyeing.

Embodiment 8

Use WT1 _332-347 Peptide activation E04.1 cell

To be similar to the method WT1 of embodiment 4 _332-347Peptide stimulates preparation from the dendritic cell of the positive healthy volunteer's blood of HLA-DRB1*0405-, with the E04.1 cytomixis, cultivates 24 hours.As negative control group, dendritic cell and the E04.1 cytomixis that will stimulate without peptide.Cultivate after 24 hours, adding final concentration is that brefeldin (Brefeldin) A of 10 μ g/ml is with the exocytosis of inhibition E04.1 cell.In addition, reclaim the CD4 positive T cell after cultivating again 6 hours, and fix with the PBC that contains 2% formaldehyde, with the cell membrane permeability of the perviousness solution-treated that contains 0.1% saponin with increase antibody.Then, by with the anti-IFN-gamma antibodies (BD, PharMingen) of PE-mark and FITC-mark anti--IL-4 antibody (BD, PharMingen) carries out dyeing in born of the same parents to handled cell, and the use flow cytometer is analyzed.The results are shown in Fig. 9.Disclosed the E04.1 cell, when with use WT1 _332-347When the dendritic cell that peptide stimulates were cultivated altogether, induced strong had produced Th-1 cytokines IFN-γ, but does not produce Th-2 cytokines IL-4.

By use anti-CD 4 antibodies and the E04.1 cell of anti--CXCR3 antibody staining without stimulation, and analyze by flow cytometer.The results are shown in Figure 10.The E04.1 cell that has disclosed over 90% is Th-1 type CD4 positive T cell, and is positive to CD4 and CXCR3.Known CXCR3 be a kind of on Th-1 type immunocyte the Chemokine Receptors of high expression level.

The above results shows WT1 _332-347Peptide activation WT1 _332-347Specific C D4 positive cell line E04.1 cell, and inducing cell produces Th-1 cytokines IFN-γ.These results show WT1 _332-347Peptide activates and makes the CD4 positive T cell to be divided into the Th-1 type.

Embodiment 9

Pass through WT1 _332-347 Peptide strengthens inducing and activating of WT1-specific CTL s

Use for same healthy volunteer (HLA-A*2402/1101, the Drb1*0405.0803) blood of setting up the E04.1 cell with the method that is similar to embodiment 4 to prepare PBMCs, and with 3 * 10 ⁴Cells/well is seeded in 24 hole culture plates.Add WT1 as follows in the hole _235-243Peptide (SEQ ID NO:27) and E04.1 cell, and at 37 ℃ of lower culture plates 7 days, i.e. WT1 _235-243Peptide (20 μ g/ml); WT1 _235-243Peptide (20 μ g/ml)+WT1 _332-347Peptide (20 μ g/ml); WT1 _235-243Peptide (20 μ g/ml)+E04.1 cell (1.5 * 10 ⁶Cells/well); Or WT1 _235-243Peptide (20 μ g/ml)+WT1 _332-347Peptide (20 μ g/ml)+E04.1 cell (1.5 * 10 ⁶Cells/well).As for substratum, use the X-VIVO15 that contains 10%AB serum ^TMSubstratum.WT1 as used herein _235-243Peptide is a kind of cancer antigen peptide (WO2004/024175) of inducing HLA-A*2402-Restricted CTL s activity that has.

Cultivate after 7 days, reclaim cell and use anti--CD8 antibody (BD PharMingen) and WT1 _235-243Dye half cell of the tetramer of peptide/HLA-A*2402 specificity PE-mark is analyzed by flow cytometer.The results are shown in Figure 11 (A-D).Already reported and used WT1 _235-243Peptide stimulates PBMCs to produce CD8 and WT1 _235-243The WT1 of peptide/HLA-A*2402 positive _235-243The inducing of peptide specific CTL precursor (Cancer ImmunolImmunother, 51, p614-620 (2002)).When using WT1 _235-243When peptide stimulates PBMCs separately, WT1 _235-243The per-cent of peptide specific CTL precursor is 0.12% (Figure 11-A).When using WT1 _235-243Peptide adds WT1 _332-347When peptide stimulates, WT1 _235-243The per-cent of peptide specific CTL precursor is increased to 0.69% (Figure 11-B).When using WT1 _235-243When peptide adds the E04.1 cell and stimulates, WT1 _235-243The per-cent of peptide specific CTL precursor further is increased to 4.51% (Figure 11-C).When using WT1 _235-243Peptide adds WT1 _332-347When peptide adds the E04.1 cell and stimulates, WT1 _235-243The per-cent of peptide specific CTL precursor is increased to again 7.12% (Figure 11-D).

Cell (3 * 10 with second half recovery ⁵Cell) with use WT1 _235-243Peptide stimulates and the dendritic cell that stand x-ray irradiation (30Gy) were cultivated 6 hours.Cultivate beginning after 1 hour, adding brefeldin A to suppress the exocytosis of cell.Cultivated lasting 5 hours again, and with resisting-CD8 antibody and WT1 _235-243The tetramer of peptide/HLA-A*2402-specificity PE-mark is to cell dyeing.Fixed cell and with the perviousness solution-treated increasing cell membrane permeability, carry out dyeing in born of the same parents with the method that the is similar to embodiment 8 anti-IFN-gamma antibodies with the PE-mark.As negative control group, with resisting-mouse IgG antibody (BDPharMingen) staining cell of APC-mark, and be all that positive cell mass is excluded as unspecific staining to IFN-γ and mouse IgG.

The results are shown in Figure 12 (A-D).When using WT1 _235-243Peptide stimulates WT1 _235-243Peptide specific CTL precursor is in the time of 6 hours, induced and to CD8, WT1 _235-243Peptide/HLA-A*2402 is positive and IFN-γ is the positive, WT1 that be specific to activation _235-243The CTLs of peptide.When using WT1 _235-243When peptide stimulates separately, be specific to the WT1 of activation _235-243The per-cent of the CTLs of peptide is 17.0% (Figure 12-A).When using WT1 _235-243Peptide adds WT1 _332-347When peptide stimulates, be specific to the WT1 of activation _235-243The per-cent of the CTLs of peptide is increased to 23.3% (Figure 12-B).When using WT1 _235-243When peptide adds the E04.1 cytositimulation, be specific to the WT1 of activation _235-243The per-cent of the CTLs of peptide is increased to 25.7% (Figure 12-C).When using WT1 _235-243Peptide adds WT1 _332-347When peptide adds the E04.1 cytositimulation, be specific to the WT1 of activation _235-243The per-cent of the CTLs of peptide is increased to 39.0% (Figure 12-D).

From the above results, disclosed WT1 _332-347Peptide is the auxiliary peptide that a kind of WT1 of enhancing specific CTL precursor is induced and activated.Also having disclosed the E04.1 cell is a kind of helper T cell of the WT1 of enhancing specific CTL s activation, and its subsidiary function is passed through WT1 _332-347Peptide is increased, and has strengthened WT1 specific CTL s activation.

Embodiment 10

WT1 _332-347 The non-kind of peptide is the research of selectivity characrerisitic

Research WT1 _332-347Peptide whether also can with it is said that the HLA-DRB1*1502 molecule that many Japaneses have is combined, and be that the auxiliary peptide of selectivity is to WT1 as non-kind _332-347Inducing of specific C D4 positive T cell.

1. experimental technique

1) preparation dendritic cell (DC)

Peripheral blood lymphocytes (PBMCs) is separated in the peripheral blood of healthy volunteer (HLA-DRB1*1502/1403), and with 1 * 10 ⁷Cells/well is seeded in uses 1%AB-type serum (Nabi, Miami, FL) and X-VIVO 15 ^TMIn 6 hole plastic boards of substratum (Cambrex), cultivated 2 hours.After removing not attached cell, containing 1000IU/ml IL-4 (PeproTech), 1000IU/ml GM-CSF (PeproTech), 1%AB type serum and X-VIVO 15 ^TMSubstratum in cultivate remaining attached cell.At the 2nd and 4 day, replaced medium also added IL-4 and GM-CSF, at the 6th day, added TNF-α to 100IU/ml so that dendritic cell maturation.

2) induce WT1 _332-347Specific C D4 positive T cell

Use for separating of the RosetteSep (StemCell) of CD4 positive T cell from same volunteer's blood separation CD4 positive T cell.With the CD4 positive T cell that obtains with 3 * 10 ⁶Cells/well is seeded in 24 hole flat boards, and uses through WT1 _332-347Peptide (20 μ g/ml) stimulate and stand radiation exposure (25Gy) from body dendritic cell (3 * 10 ⁵Cell) stimulate.Stimulate the 2nd day, add IL-2 to 20IU/ml.With similar method, use through WT1 _332-347The dendritic cell that peptide (20 μ g/ml) stimulates sting CD4 positive T cell that regular menstruation during early pregnancy stimulates once week about.In addition, use after stimulating for the second time and contain the IL-2 substratum every other day replaced medium is once.In this experiment, the CD4 positive T cell of inducing stimulates through 3 times altogether.

3) growth measurement

By [ ³H]-the thymidine method of mixing carries out growth measurement.Peptide is stimulated the CD4 positive T cell (3 * 10 of inducing ⁴Cell; Effector) with being selected from WT1 _332-347, WT1 _172-186And WT1 _225-243The peptide of peptide stimulates and stands the PBMCs (1 * 10 of radiation exposure ⁵Cell; " stimulator ") cultivate altogether in 96 hole flat boards.As negative control, use the DC (-) that stimulates without peptide.After cultivating altogether in 80 hours, with the 37kBq/ hole add [ ³H]-thymidine (AmershamBiosciences).This flat board incubation was also measured with the β scintillometer again in 16 hours.Linear module is " counting/minute (cpm) ", and each mensuration repeats three times.

4) produce by the flow cytometry analysis cytokine

With the identical method of growth measurement, CD4 positive T cell and stimulator are cultivated 2 hours altogether, and add wherein brefeldin A.After 4 hours, reclaim cell, be fixed and osmotic treated, with the FITC-mark anti--IL-4 antibody (BD Pharmingen) and PE-mark anti--IFN-gamma antibodies (BD Pharmingen) dyes, analyzes by flow cytometer.

5) ELISA measures

By radiation exposure (25Gy) use or without WT1 _332-347The PBMCs (6 * 10 that peptide stimulates ⁵Cell), every group of cell and WT1 _332-347The CD4 positive T cell (6 * 10 of inducing ⁵Cell) cultivate altogether.After cultivating in 72 hours, reclaim supernatant liquor, 300 μ l solution are used for IL-4 and IFN-γ mensuration.

6) the TCR response spectrum is measured

Use TCR V beta response spectrum test kit (BECKMAN COULTER), FACsort (BECTON DICKINSON) to WT1 _332-347The response spectrum of T-cell receptors (TCR) the β chain of the CD4 positive T cell of inducing peptide is analyzed.

2. experimental result

Separation is used through WT1 from the CD4 positive T cell of healthy volunteer (HLA-DRB1*1502/1403) blood _332-347What peptide stimulated stimulates 3 times altogether from the body dendritic cell.By using respectively WT1 _172-186, WT1 _225-243And WT1 _332-347The growth measurement of peptide, the specificity of the CD4 positive T cell that institute induces to this peptide.As a result, WT1 _332-347The CD4 positive T cell of inducing peptide is lacking this peptide or is using WT1 _172-186Or WT1 _225-243Can't breed under the condition that stimulates, but when using WT1 _332-347During stimulation, propagation reaches approximately 10 times more than (Figure 13).According to these results, the CD4 positive T cell of inducing is to WT1 _332-347Has specificity.

WT1 _332-347The CD4 positive T cell of inducing peptide carries out the TCR response spectrum and measures.The results are shown in Figure 14.Have the cell of V β 3 and have a V β 20 those preponderate, account for respectively 10% of sum.

These dominant CD4 positive T cells separate by sorting, have the clone called after E15.1 subbreed of V β 3, and have the called after E15.2 subbreed of V β 20.In these two kinds of clones, the E15.2 subbreed has shown WT1 _332-347Peptide is higher replys (Figure 15).

Then, use WT1 _332-347Peptide stimulates the E15.2 subbreed, measures secreted IL-4 and IFN-γ by Intracellular cytokine staining methods.As a result, disclosed IFN-γ (Th-1 cytokines) but not IL-4 (Th-2 cytokines) preponderates (Figure 16) in the cytokine that described clone produces.Also disclosed the WT1 of E15.2 subbreed _332-347Proliferated specifically is replied and is depended on WT1 _332-347Concentration (Figure 17).These results show that the E15.2 subbreed is a kind of WT1 of being specific to _332-347Th-1 type CD4 positive T cell system.

As mentioned above, might be from positive to the HLA-DRB1*1502 molecule, but being induced, the CD4 positive T cell in the healthy volunteer source of HLA-DRB1*0405 molecule feminine gender is specific to WT1 _332-347Th-1 type CD4 positive T cell system.Based on these results, think that described CD4 positive T cell participates in cellular immunity and can activate CTLs by secrete cytokines.Therefore, by being used in combination WT1 _332-347With the restricted WT1 peptide of the HLA-I class that can activate CTLs (cancer antigen peptide), shown that anti-tumour effect can further be enhanced.

Use WT1 _332-347Peptide stimulates PBMCs from the positive healthy volunteer (1502/0901) of HLA-DRB1*1502 or the negative healthy volunteer of HLA-DRB1*1502 (1302/0803) to obtain stimulator.Stimulator is cultivated altogether with the E15.2 subbreed respectively, and by the growth measurement analysis to WT1 _332-347Specific propagation.The results are shown in Figure 18.In the healthy volunteer of the HLA-DRB1*1502-positive, observe WT1 _332-347Proliferated specifically, but in the healthy volunteer of HLA-DRB1*1502-feminine gender, do not observe propagation (Figure 18).This shows the WT1 of E15.2 subbreed _332-347Proliferated specifically is limited to HLA-DRB1*1502.

As mentioned above, use is specific to WT1 _332-347The E15.2 subbreed that the Th-1 type CD4 positive T cell of peptide is is analyzed, and shows WT1 _332-347Be that a kind of non-kind is the auxiliary peptide of selectivity, it not only is combined with the HLA-DRB1*0405 molecule, also is combined with the HLA-DRB1*1502 molecule, and these molecules frequency of occurrences in the Japanese is respectively the first and the 3rd.

Industrial applicibility

The invention provides the derivative HLA-DRB1*0405 associativity antigen peptide of a kind of WT1, the polynucleotide of coding for said peptides comprise helper T cell inductor of described peptide or polynucleotide etc.Helper T cell inductor of the present invention is used as the toughener of cancer vaccine effect.Cancer vaccine effect toughener of the present invention especially is used as WT1 efficacy of vaccines toughener applicable to the cancer patients of many HLA-DRB1*0405 positives.

Sequence table

<110>Haruo，Sugiyama

＜120〉the derivative HLA-DR conjugated antigen peptide of WT1

<130>

<140>2003-375603

<141>2003-11-05

<160>56

<170>PatentIn Ver.2.1

<210>1

<211>449

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>1

Met Gly Ser Asp Val Arg Asp Leu Asn Ala Leu Leu Pro Ala Val Pro

1 5 10 15

Ser Leu Gly Gly Gly Gly Gly Cys Ala Leu Pro Val Ser Gly Ala Ala

20 25 30

Gln Trp Ala Pro Val Leu Asp Phe Ala Pro Pro Gly Ala Ser Ala Tyr

35 40 45

Gly Ser Leu Gly Gly Pro Ala Pro Pro Pro Ala Pro Pro Pro Pro Pro

50 55 60

Pro Pro Pro Pro His Ser Phe Ile Lys Gln Glu Pro Ser Trp Gly Gly

65 70 75 80

Ala Glu Pro His Glu Glu Gln Cys Leu Ser Ala Phe Thr Val His Phe

85 90 95

Ser Gly Gln Phe Thr Gly Thr Ala Gly Ala Cys Arg Tyr Gly Pro Phe

100 105 110

Gly Pro Pro Pro Pro Ser Gln Ala Ser Ser Gly Gln Ala Arg Met Phe

115 120 125

Pro Asn Ala Pro Tyr Leu Pro Ser Cys Leu Glu Ser Gln Pro Ala Ile

130 135 140

Arg Asn Gln Gly Tyr Ser Thr Val Thr Phe Asp Gly Thr Pro Ser Tyr

145 150 155 160

Gly His Thr Pro Ser His His Ala Ala Gln Phe Pro Asn His Ser Phe

165 170 175

Lys His Glu Asp Pro Met Gly Gln Gln Gly Ser Leu Gly Glu Gln Gln

180 185 190

Tyr Ser Val Pro Pro Pro Val Tyr Gly Cys His Thr Pro Thr Asp Ser

195 200 205

Cys Thr Gly Ser Gln Ala Leu Leu Leu Arg Thr Pro Tyr Ser Ser Asp

210 215 220

Asn Leu Tyr Gln Met Thr Ser Gln Leu Glu Cys Met Thr Trp Asn Gln

225 230 235 240

Met Asn Leu Gly Ala Thr Leu Lys Gly Val Ala Ala Gly Ser Ser Ser

245 250 255

Ser Val Lys Trp Thr Glu Gly Gln Ser Asn His Ser Thr Gly Tyr Glu

260 265 270

Ser Asp Asn His Thr Thr Pro Ile Leu Cys Gly Ala Gln Tyr Arg Ile

275 280 285

His Thr His Gly Val Phe Arg Gly Ile Gln Asp Val Arg Arg Val Pro

290 295 300

Gly Val Ala Pro Thr Leu Val Arg Ser Ala Ser Glu Thr Ser Glu Lys

305 310 315 320

Arg Pro Phe Met Cys Ala Tyr Pro Gly Cys Asn Lys Arg Tyr Phe Lys

325 330 335

Leu Ser His Leu Gln Met His Ser Arg Lys His Thr Gly Glu Lys Pro

340 345 350

Tyr Gln Cys Asp Phe Lys Asp Cys Glu Arg Arg Phe Ser Arg Ser Asp

355 360 365

Gln Leu Lys Arg His Gln Arg Arg His Thr Gly Val Lys Pro Phe Gln

370 375 380

Cys Lys Thr Cys Gln Arg Lys Phe Ser Arg Ser Asp His Leu Lys Thr

385 390 395 400

His Thr Arg Thr His Thr Gly Lys Thr Ser Glu Lys Pro Phe Ser Cys

405 410 415

Arg Trp Pro Ser Cys Gln Lys Lys Phe Ala Arg Ser Asp Glu Leu Val

420 425 430

Arg His His Asn Met His Gln Arg Asn Met Thr Lys Leu Gln Leu Ala

435 440 445

Leu

<210>2

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

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Leu Val Arg His His Asn Met His Gln

1 5

<210>3

<211>9

<212>PRT

＜213〉artificial sequence

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＜223〉artificial sequence description: synthetic peptide

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Leu Tyr Gln Met Thr Ser Gln Leu Glu

1 5

<210>4

<211>9

<212>PRT

＜213〉artificial sequence

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＜223〉artificial sequence description: synthetic peptide

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Phe Lys His Glu Asp Pro Met Gly Gln

1 5

<210>5

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>5

Leu Val Arg Ser Ala Ser Glu Thr Ser

1 5

<210>6

<211>9

<212>PRT

＜213〉artificial sequence

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＜223〉artificial sequence description: synthetic peptide

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Met Gly Gln Gln Gly Ser Leu Gly Glu

1 5

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<212>PRT

＜213〉artificial sequence

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＜223〉artificial sequence description: synthetic peptide

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Val Tyr Gly Cys His Thr Pro Thr Asp

1 5

<210>8

<211>9

<212>PRT

＜213〉artificial sequence

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＜223〉artificial sequence description: synthetic peptide

<400>8

Leu Arg Thr Pro Tyr Ser Ser Asp Asn

1 5

<210>9

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>9

Phe Ile Lys Gln Glu Pro Ser Trp Gly

1 5

<210>10

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>10

Trp Gly Gly Ala Glu Pro His Glu Glu

1 5

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<211>9

<212>PRT

＜213〉artificial sequence

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＜223〉artificial sequence description: synthetic peptide

<400>11

Phe Lys Leu Ser His Leu Gln Met His

1 5

<210>12

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>12

Tyr Phe Lys Leu Ser His Leu Gln Met

1 5

<210>13

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>13

Leu Glu Cys Met Thr Trp Asn Gln Met

1 5

<210>14

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>14

Phe Arg Gly Ile Gln Asp Val Arg Arg

1 5

<210>15

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>15

Leu Leu Pro Ala Val Pro Ser Leu Gly

1 5

<210>16

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>16

Leu Ser Ala Phe Thr Val His Phe Ser

1 5

<210>17

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>17

Met Asn Leu Gly Ala Thr Leu Lys Gly

1 5

<210>18

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>18

Val Arg Ser Ala Ser Glu Thr Ser Glu

1 5

<210>19

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>19

Leu Pro Ala Val Pro Ser Leu Gly Gly

1 5

<210>20

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>20

Tyr Gly Cys His Thr Pro Thr Asp Ser

1 5

<210>21

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>21

Phe Ser Gly Gln Phe Thr Gly Thr Ala

1 5

<210>22

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>22

Phe Met Cys Ala Tyr Pro Gly Cys Asn

1 5

<210>23

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>23

Tyr Gln Met Thr Ser Gln Leu Glu Cys

1 5

<210>24

<211>16

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>24

Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His

1 5 10 15

<210>25

<211>15

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>25

Pro Asn His Ser Phe Lys His Glu Asp Pro Met Gly Gln Gln Gly

1 5 10 15

<210>26

<211>19

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>26

Asn Leu Tyr Gln Met Thr Ser Gln Leu Glu Cys Met Thr Trp Asn Gln

1 5 10 15

Met Ash Leu

<210>27

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>27

Cys Met Thr Trp Asn Gln Met Asn Leu

1 5

<210>28

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>28

Cys Tyr Thr Trp Asn Gln Met Asn Leu

1 5

<210>29

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>29

Arg Met Phe Pro Asn Ala Pro Tyr Leu

1 5

<210>30

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>30

Arg Tyr Pro Ser Cys Gln Lys Lys Phe

1 5

<210>31

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>31

Ser Tyr Thr Trp Asn Gln Met Asn Leu

1 5

<210>32

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>32

Ala Tyr Thr Trp Asn Gln Met Asn Leu

1 5

<210>33

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<223>Xaa at 1 position stands for Abu.

<400>33

Xaa Tyr Thr Trp Asn Gln Met Asn Leu

1 5

<210>34

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>34

Arg Tyr Thr Trp Asn Gln Met Asn Leu

1 5

<210>35

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>35

Lys Tyr Thr Trp Asn Gln Met Asn Leu

1 5

<210>36

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>36

Arg Tyr Phe Pro Asn Ala Pro Tyr Leu

1 5

<210>37

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>37

Arg Tyr Pro Gly Val Ala Pro Thr Leu

1 5

<210>38

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>38

Ala Tyr Leu Pro Ala Val Pro Ser Leu

1 5

<210>39

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>39

Asn Tyr Met Asn Leu Gly Ala Thr Leu

1 5

<210>40

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>40

Arg Val Pro Gly Val Ala Pro Thr Leu

1 5

<210>41

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>41

Arg Tyr Pro Ser Ser Gln Lys Lys Phe

1 5

<210>42

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>42

Arg Tyr Pro Ser Ala Gln Lys Lys Phe

1 5

<210>43

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<223>Xaa at 5 position stands for Abu.

<400>43

Arg Tyr Pro Ser Xaa Gln Lys Lys Phe

1 5

<210>44

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>44

Ser Leu Gly Glu Gln Gln Tyr Ser Val

1 5

<210>45

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>45

Asp Leu Asn Ala Leu Leu Pro Ala Val

1 5

<210>46

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>46

Tyr Arg Ile His Thr His Gly Val Phe

1 5

<210>47

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>47

Leu Val Arg His His Asn Met His Gln

1 5

<210>48

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>48

Tyr Gln Met Thr Ser Gln Leu Glu Cys

1 5

<210>49

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>49

Leu Gln Met His Ser Arg Lys His Thr

1 5

<210>50

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>50

Tyr Phe Lys Leu Ser His Leu Gln Met

1 5

<210>51

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>51

Val Lys Pro Phe Gln Cys Lys Thr Cys

1 5

<210>52

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>52

Leu Lys Arg His Gln Arg Arg His Thr

1 5

<210>53

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>53

Leu Lys Thr His Thr Arg Thr His Thr

1 5

<210>54

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>54

Tyr Gly Pro Phe Gly Pro Pro Pro Pro

1 5

<210>55

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>55

Val Arg His His Asn Met His Gln Arg

1 5

<210>56

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉artificial sequence description: synthetic peptide

<400>56

Phe Pro Asn Ala Pro Tyr Leu Pro Ser

1 5

Claims

1. the purposes of the peptide of aminoacid sequence as shown in SEQ ID NO:24 in the patient's who has HLA DRB1*1502 for the preparation for the treatment of or prevention the pharmaceutical composition of cancer.

2. the purposes of claim 1, wherein the patient has HLA DRB1*1502 and HLA DRB1*0405.

3. the peptide purposes in pharmaceutical compositions of aminoacid sequence as shown in SEQ ID NO:24, described pharmaceutical composition are the inductors with helper cell in the patient of HLA DRB1*1502.

4. the purposes of claim 3, wherein the patient has HLA DRB1*1502 and HLA DRB1*0405.

5. the peptide purposes in pharmaceutical compositions of aminoacid sequence as shown in SEQ ID NO:24, described pharmaceutical composition are the enhansers with cancer vaccine validity in the patient of HLA DRB1*1502.

6. the purposes of claim 5, wherein the patient has HLA DRB1*1502 and HLA DRB1*0405.

7. claim 5 or 6 purposes, wherein said cancer vaccine is the cancer antigen peptide vaccine.